Cell permeable Nm23 recombinant proteins, polynucleotides encoding the same, and anti-metastatic composition comprising the same

ABSTRACT

The present invention discloses cell permeable Nm23 recombinant proteins where a macromolecule transduction domain (MTD) is fused to a metastasis suppressor Nm23. Also disclosed are polynucleotides encoding the cell permeable Nm23 recombinant proteins, an expression vector containing the cell permeable Nm23 recombinant protein, and a pharmaceutical composition for preventing metastasis which contains the cell permeable Nm23 recombinant protein as an effective ingredient. The cell permeable Nm23 recombinant proteins of the present invention can induce KSR phosphorylation and inactivation and inhibit Ras-mediated MAPK cascade by efficiently introducing a metastasis suppressor Nm23 into a cell. Therefore, the cell permeable Nm23 recombinant proteins of the present invention can be effectively used as an anti-metastatic agent capable of preventing cancer metastasis by inhibiting the proliferation, differentiation and migration of cancer cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of PCT/KR08/005221 filed Sep. 4, 2008 and claims the benefit of U.S. 60/969,714 filed Sep. 4, 2007.

TECHNICAL FIELD

The present invention relates to cell permeable Nm23 recombinant proteins in which a metastasis suppressor Nm23 is fused to a macromolecule transduction domain (MTD), polynucleotides encoding the same, expression vectors for producing the same, and anti-metastatic pharmaceutical compositions including the same as effective ingredients for inhibiting metastasis.

BACKGROUND ART

Nm23 gene has been reported to encode proteins involved in the development and differentiation of a normal tissue, and its expression is decreased in various metastatic cells. Nm23 proteins belong to a tumor metastasis suppressor and generally consist of 150 to 180 amino acids. Nm23 proteins contain a leucine zipper motif and exhibit nucleoside diphosphate kinase (NDPK) activity. Two human Nm23 homologues, Nm23-H1 and Nm23-H2, consist of 152 amino acids having molecular weights of 17,143 and 17,294, respectively. In particular, it has been found that Nm23-H1 plays an important role in tumor metastasis and other various cell mechanisms including cell proliferation, embryogenesis, differentiation and tumorigenesis.

The mechanisms by which Nm23 affects tumor development and metastasis have not yet been clearly investigated. NDPK transfers a phosphoryl group between nucleoside triphosphate and nucleoside diphosphate via a covalent phosphoenzyme intermediate. For such a phosphorylation, histidine 118 of each Nm23-H1 and Nm23-H2 is served as a target site. Apart from the NDPK-mediated histidine phosphorylation, serine autophosphorylation was observed in Nm23 (MacDonald N J et al., J. Biol. Chem. 268: 25780-25789, 1993). When melanoma cells of Nm23 transfected mice were compared with a control cell, there was a direct correlation between the in vivo phosphorylation level of Nm23 serine and the inhibition of tumor metastatic potential. Serine phosphorylation of mouse Nm23 is inhibited by cAMP in vivo, while by forskolin in vitro, which demonstrates that the phosphorylation is controlled by signal transduction pathways.

Initially, Nm23 expression was reported to closely correlate to mouse melanoma cell lines with poor metastatic potential. The relationship between Nm23 reduced expression and tumor metastasis has been regarded as direct evidence to support the fact that Nm23 functions as a tumor metastasis suppressor (Steeg, P. S., Breast Dis. 10: 47-50, 1998). Inducible overexpression of Nm23 exhibited remarkably reduced metastatic potential in a highly metastatic cancer cell line. The Nm23 gene cloned as a putative tumor metastasis suppressor gene exhibits serine/threonine specific phosphotransferase and histidine protein kinase activities, as well as NDPK activity. Further, the expression of Nm23 is reduced as hematopoietic stem cells (HSC) are differentiated, which suggests that Nm23 is an important factor for anti-differentiation in those cells (Gervasi, F. et al., Cell Growth Differ 7: 1689-95, 1996). It has been found that Nm23 exhibits a strong inhibitory effect on tumor metastasis upon temporary transfection in forced inducible gene expression and in vitro metastasis model systems of human tumors (Hirayama, R. et al., J. Natl. Cancer Inst. 83: 1249-50, 1991; Nakayama, T. et al., J. Natl. Cancer Inst. 84: 1349-54, 1992; Leone, A. et al., Oncogene 8: 2325-33, 1993; Leone, A. et al., Oncogene 8: 855-65, 1993). In contrast, Nm23 mutation, leading to the loss of NDPK activity, had no influence on the inhibitory function of Nm23 in breast cancer cells (MacDonald, N. J. et al., J. Biol. Chem. 271:25107-16, 1996).

The most authentic evidence for the fact that Nm23 is a metastasis suppressor is revealed when the Nm23 gene is transfected into tumor cell lines. In metastatically competent cells, the administration of high dose Nm23 showed reduced metastatic activity by 40 to 98% as compared with a control transfectant (Leone, A. et al, Cell 65: 25-35, 1991; Leone, A. et al., Oncogene 8:2325-33, 1993).

Recently, it has been reported that Nm23 interacts with a kinase suppressor of Ras (KSR) discovered in the Drosophilae (Drosophilar melanogaster) and nematode (Caenorhabditis elegans) systems (Morrison, D. K., J. Cell Sci. 114: 1609-12, 2001). KSR is a scaffold protein of a mitogen-activated protein kinase (MAPK) cascade (Burack, W. R. and Shaw, A. S., Curr. Opin. Cell Biol. 12: 211-6, 2000; Pawson, T. and Scott, J. D., Science 278: 2075-80, 1997). Such scaffold protein is necessary to enhance the rate of phosphorylation and contribute to the specificity and stabilization of the phosphorylation pathway. Once the MAPK signal transduction pathway is activated by active Ras, KSR is forcedly dephosphorylated, and then, serve as a scaffold for the activation of MAPK cascade. During this process, Nm23 phosphorylates KSR serine 392, which is a binding site for another associated protein of KSR. If the serine 392 is mutated, Nm23 phosphorylates KSR serine 434. The metastatic inhibitory activity of Nm23 has been clearly demonstrated by the fact that metastatic potential is inhibited in various tumor cells transfected with Nm23 gene (Yoshida, T. et al., J Gastroenterol. 35: 768-74, 2000). In cells activated by the stimulation of the MAPK cascade, the interaction between Nm23 and KSR induces KSR phosphorylation in vitro in a complicated manner via a histidine-dependent pathway (Hartsough, M. T. et al., J. Biol. Chem. 277: 32389-99, 2002). Further, the in vivo association of KSR and Nm23 inhibits the phenotypic effect of active Ras which activates the MAPK cascade.

Accordingly, the administration of high dose Nm23 protein may phosphorylate and inactivate KSR in vivo, leading to the inhibition of Ras-mediated MAPK cascade. The present inventors have therefore believed that the inhibition of MAPK signal transduction pathway mediated by re-phosphorylation of KSR may inhibit cell proliferation, differentiation and migration of cancer cells and exhibit anti-metastatic effect in various human cancers, and endeavored to develop new anti-metastatic agents by using the Nm23 protein.

Meanwhile, small molecules derived from synthetic compounds or natural compounds can be transported into the cells, whereas macromolecules, such as proteins, peptides, and nucleic acids, cannot. It is widely understood that macromolecules larger than 500 kDa are incapable of penetrating the plasma membrane, i.e., the lipid bilayer structure, of live cells. To overcome this problem, a macromolecule intracellular transduction technology (MITT) was developed (Jo et al., Nat. Biotech. 19: 929-33, 2001), which allows the delivery of therapeutically effective macromolecules into cells, making the development of new drugs using peptides, proteins and genetic materials possible. According to this method, if a target macromolecule is fused to a hydrophobic macromolecule transduction domain (MTD) and other cellular delivery regulators, synthesized, expressed, and purified in the form of a recombinant protein, it can penetrate the plasma membrane lipid bilayer of the cells, be accurately delivered to a target site, and then, effectively exhibit its therapeutic effect. Such MTDs facilitate the transport of many impermeable materials which are fused to peptides, proteins, DNA, RNA, synthetic compounds, and the like into the cells.

Accordingly, the inventors of the present invention have developed a method of mediating the transport of a metastasis suppressor Nm23 into the cells, where cell permeable Nm23 recombinant proteins are engineered by fusing a MTD to the metastasis suppressor Nm23. Such cell permeable Nm23 recombinant proteins have been found to efficiently mediate the transport of a metastasis suppressor Nm23 into the cells in vivo as well as in vitro and can be used as anti-metastatic agents for inhibiting metastasis occurring in various human cancers.

DISCLOSURE Technical Problem

Accordingly, the objective of the present invention is to provide cell permeable Nm23 recombinant proteins as an anti-metastatic agent which is effective for preventing metastasis in various kinds of human cancers by inhibiting proliferation, differentiation and migration of cancer cells.

Technical Solution

One aspect of the present invention relates to cell permeable Nm23 recombinant proteins capable of mediating the transport of a metastasis suppressor Nm23 into a cell by fusing a macromolecule transduction domain (MTD) having cell permeability to the metastasis suppressor protein.

Another aspect of the present invention relates to polynucleotides encoding the above cell permeable Nm23 recombinant proteins.

The present invention also relates to expression vectors containing the above polynucleotides, and transformants transformed with the above expression vectors.

Another aspect of the present invention relates to a method of producing cell permeable Nm23 recombinant proteins involving culturing the above transformants.

Another aspect of the present invention relates to a pharmaceutical composition including the above cell permeable Nm23 recombinant proteins as an effective ingredient for inhibiting metastasis.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram illustrating the structures of cell permeable Nm23 recombinant proteins being fused to one of a kFGF4-derived MTD, JO-76 MTD and JO-77 MTD and constructed in the full-length forms according to the present invention.

FIG. 2 a is a photograph of an agarose gel electrophoresis analysis showing PCR-amplified DNA fragments encoding cell permeable Nm23 recombinant proteins being fused to a kFGF4-derived MTD and constructed in the full-length forms according to the present invention.

FIG. 2 b is a photograph of an agarose gel electrophoresis analysis showing PCR-amplified DNA fragments encoding cell permeable Nm23 recombinant proteins being fused to each of JO-76 and JO-77 MTDs and constructed in the full-length forms according to the present invention.

FIG. 3 a is a schematic diagram illustrating the subcloning of a PCR product encoding a cell permeable Nm23 recombinant protein into the pGEM-T Easy vector according to the present invention.

FIGS. 3 b and 3 c are photographs of an agarose gel electrophoresis analysis showing the PCR products encoding the cell permeable Nm23 recombinant proteins from FIGS. 2 a and 2 b subcloned in the pGEM-T Easy vector according to the present invention, respectively.

FIG. 4 a is a schematic diagram illustrating the cloning of a recombinant DNA fragment encoding a cell permeable Nm23 recombinant protein into the pET 28(+) vector according to the present invention.

FIGS. 4 b and 4 c are photographs of an agarose gel electrophoresis analysis showing the recombinant DNA fragments encoding the cell permeable Nm23 recombinant proteins subcloned in the pET 28(+) vector according to the present invention.

FIG. 5 is a photograph of a SDS-PAGE analysis showing the inducible expression of cell permeable Nm23 recombinant proteins according to the present invention in various kinds of host cells.

FIG. 6 is a photograph of a SDS-PAGE analysis showing the purification of cell permeable Nm23 recombinant proteins expressed from the transformants where the expression vector according to the present invention is transformed into.

FIGS. 7 a and 7 b are graphs illustrating the results of flow cytometry analysis of cell permeabilities of cell permeable Nm23 recombinant proteins according to the present invention.

FIGS. 8 a to 8 c are confocal laser scanning microscopy photographs visualizing the cell permeabilities of cell permeable Nm23 recombinant proteins according to the present invention in mouse fibroblasts.

FIGS. 9 a and 9 b are photographs of a Western blot analysis showing the inhibitory effect of cell permeable Nm23 recombinant proteins according to the present invention on MAPK signal transduction.

FIGS. 10 a and 10 b are photographs of an invasion analysis showing the inhibitory effect of cell permeable Nm23 recombinant proteins according to the present invention on metastasis.

FIG. 11 is a photograph of a wound migration assay showing the inhibitory effect of cell permeable Nm23 recombinant proteins according to the present invention on metastasis.

FIG. 12 a is a photograph illustrating the inhibitory effect on metastasis in a mouse lung tissue extracted from a mouse administered with the cell permeable Nm23 recombinant protein according to the present invention.

FIG. 12 b is a photograph of immunohistochemical staining showing the expression of a metastatic marker, vimentin, in a mouse lung tissue extracted from a mouse administered with the cell permeable Nm23 recombinant protein according to the present invention.

FIG. 13 is a photograph of a terminus deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis showing the apoptosis-inducing effect in a mouse lung tissue extracted from a mouse administered with the cell permeable Nm23 recombinant protein according to the present invention.

FIG. 14 is a photograph of a microarray analysis showing differential gene expression in a mouse lung tissue extracted from a mouse administered with the cell permeable Nm23 recombinant protein according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides cell permeable Nm23 recombinant proteins (CP-Nm23) capable of mediating the transport of a metastasis suppressor Nm23 into a cell in which the metastasis suppressor Nm23 is fused to a macromolecule transduction domain and, thereby, imparted with cell permeability; and polynucleotides encoding each of the cell permeable Nm23 recombinant proteins.

The present invention is characterized in that a metastasis suppressor Nm23 which is a macromolecule incapable of being introduced into a cell is fused to a specific macromolecule transduction domain (hereinafter, “MTD”) peptide so as to provide cell permeability, and thus, can be effectively transported into a cell. The MTD peptide may be fused to the N-terminus, the C-terminus, or both termini of the metastasis suppressor Nm23.

The present invention relates to cell permeable Nm23 recombinant proteins that are engineered by fusing a metastasis suppressor Nm23 to one of three MTD domains capable of mediating the transport of a macromolecule into a cell.

The term “cell permeable Nm23 recombinant protein” as used herein refers to a covalent bond complex bearing a MTD and a metastasis suppressor protein Nm23, where they are functionally linked by genetic fusion or chemical coupling. Here, the term “genetic fusion” refers to a co-linear, covalent linkage of two or more proteins or fragments thereof via their individual peptide backbones, through genetic expression of a polynucleotide molecule encoding those proteins.

Nm23 is a metastasis suppressor protein which inhibits the proliferation, differentiation and migration of cancer cells and induces apoptosis by controlling the MAPK signal transduction cascade which is mediated by KSR phosphorylation. Nm23 has an amino acid sequence represented by SEQ ID NO: 2, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 1. Nm23 functions as an important target protein in signal transduction cascades including KSR and Ras-mediated MAPK.

It has been reported that Nm23 is an endogeneous protein and exhibits NDP (nucleotide diphosphate)-kinase enzyme activity (Biggs et al., Cell 63, 933-940, 1990). Nm23 has also been found to be a transcription factor and cell differentiation inhibitor (I factor) (Postel et al., Science 261, 478-480, 1993; Okabe-Kado et al., Biochim. Biophys. Acta. 1267, 101-106, 1995).

In humans, eight nm23 isotypes (nm23-H1, nm23-H2, DR-nm23, nm23-H4, nm23-H5, nm23-H6, nm23-H7, and nm23-H8) have been identified to date, all of which are implicated in the regulation of metastasis (Rosengard et al., Nature 342, 177-180, 1989; Charpin C. et al., Int. J. Cancer 74, 416-420, 1997). In certain embodiments, cell permeable recombinant proteins for Nm23-H1 have been constructed, but are not limited thereto.

For the MTD capable of being fused to the metastasis suppressor Nm23, cell permeable peptides having an amino acid sequence selected from the group consisting of SEQ ID NOS: 4, 6, 8, and 37 to 227 may be used. The MTD having one of the amino acid sequences represented by SEQ ID NOS: 4, 6, 8 and 37 to 227 is a cell permeable polypeptide which is capable of mediating the transport of a biologically active molecule, such as a polypeptide, a protein domain, or a full-length protein across the cell membrane. Suitable MTDs for the present invention include a hydrophobic region showing cell membrane targeting activity by forming a helix structure at a signal peptide which is composed of an N-terminal domain, a hydrophobic domain and a C-terminal domain containing a secreted protein cleavage site. These MTDs can directly penetrate the cell membrane without causing any cell damage, transport a target protein into a cell, and thus, allow the target protein to exhibit its desired function.

The MTDs having the amino acid sequences represented by SEQ ID NOS: 4, 6, 8, and 37 to 227 and capable of being fused to a metastasis suppressor Nm23 according to the present invention are summarized in the following Tables 1a to 11.

TABLE 1a MTD Origin Amino acid sequence SEQ ID NO JO-01 CAC04038 putative NLP/P60-family Ala Val Val Val Cys Ala Ile 37 secreted protein [Streptomyces Val Leu Ala Ala Pro coelicolor A3(2)] JO-02 NP_057021 phosphatidylinositol Pro Leu Ala Leu Leu Val Leu 38 glycan, class T precursor Leu Leu Leu Gly Pro [Homo sapiens] JO-03 NP_072171 chorionic Leu Leu Leu Ala Phe Ala Leu 39 somatomammotropin hormone 2 Leu Cys Leu Pro isoform 3 [Homo sapiens] JO-04 NP_932156 nudix-type motif 9 Leu Leu Gly Ala Leu Ala Ala 40 isoform a [Homo sapiens] Val Leu Leu Ala Leu Ala JO-05 NP_057327 NAD(P)H: quinone Pro Val Leu Leu Ala Leu Gly 41 oxidoreductase type 3, polypeptide A2 Val Gly Leu Val Leu Leu Gly [Homo sapiens] Leu Ala Val JO-06 CAD55300 putative secreted protein. Ala Ala Ala Ala Val Leu Leu 42 [Streptomyces coelicolor A3(2)] Ala Ala JO-07 NP_629514 secreted protein Ile Val Val Ala Val Val Val Ile 43 [Streptomyces coelicolor A3(2)] JO-08 CAB57190 putative secreted chitin Ala Val Leu Ala Pro Val Val 44 binding protein [Streptomyces Ala Val coelicolor A3(2)] JO-09 CAB51015 putative secreted protein Leu Ala Val Cys Gly Leu Pro 45 [Streptomyces coelicolor A3(2)] Val Val Ala Leu Leu Ala JO-10 NP_625021 glycosyl hydrolase (secreted Leu Gly Gly Ala Val Val Ala Ala 46 protein) [Streptomyces coelicolor A3(2)] Pro Val Ala Ala Ala Val Ala Pro JO-11 NP_630686 secreted protein Leu Leu Leu Val Leu Ala Val 47 [Streptomyces coelicolor A3(2)] Leu Leu Ala Val Leu Pro JO-12 NP_057329 dehydrogenase/reductase Leu Leu Ile Leu Leu Leu Leu 48 (SDR family) member 8 [Homo sapiens] Pro Leu Leu Ile Val JO-13 NP_639877 putative secreted protein Leu Ala Ala Ala Ala Leu Ala 49 [Streptomyces coelicolor A3(2)] Val Leu Pro Leu JO-14 NP_699201 protease inhibitor 16 Phe Leu Met Leu Leu Leu Pro 50 precursor [Homo sapiens] Leu Leu Leu Leu Leu Val Ala JO-15 NP_639871 putative secreted protein Ala Ala Ala Ala Ala Ala Leu 51 [Streptomyces coelicolor A3(2)] Gly Leu Ala Ala Ala Val Pro Ala JO-16 CAB85250 putative secreted protein Leu Leu Leu Ala Ala Leu Leu 52 [Neisseria meningitidis Z2491] Leu Ile Ala Phe Ala Ala Val

TABLE 1b MTD Origin Amino acid sequence SEQ ID NO JO-17 NP_626397 small secreted hydrophilic Ala Leu Ala Ala Val Val Leu Ile 53 protein [Streptomyces coelicolor A3(2)] Pro Leu Gly Ile Ala Ala JO-18 CAB38593 putative secreted protein Ala Ala Leu Ala Leu Gly Val Ala 54 [Streptomyces coelicolor A3(2)] Ala Ala Pro Ala Ala Ala Pro Ala JO-19 CAB57190 putative secreted chitin Ala Ala Leu Ile Gly Ala Val Leu 55 binding protein  Ala Pro Val Val Ala Val [Streptomyces coelicolor A3(2)] JO-20 NP_626007 secreted cellulose-binding Ala Ala Gly Ile Ala Val Ala Ile 56 protein [Streptomyces coelicolor A3(2)] Ala Ala Ile Val Pro Leu Ala JO-21 NP_625632 secreted protein Ile Ala Val Ala Ile Ala Ala Ile 57 [Streptomyces coelicolor A3(2)] Val Pro Leu Ala JO-22 CAC31790 putative secreted protein Val Ala Met Ala Ala Ala Ala Val 58 [Mycobacterium leprae] Leu Ala Ala Pro Ala Leu Ala JO-23 NP_630266 secreted Protein Leu Ala Val Leu Val Leu Leu Val 59 [StrePtomyces coelicolor A3(2)] Leu Leu Pro JO-24 NP_630165 secreted Protein Val Val Ala Val Leu Ala Pro Val 60 [StrePtomyces coelicolor A3(2)] Leu JO-25 NC_003888 secreted Protein Ala Ala Leu Leu Leu Pro Leu Leu 61 [StrePtomyces coelicolor A3(2)] Leu Leu Leu Pro JO-26 NP_627363 secreted Protein Pro Ala Ala Val Ala Ala Leu Leu 62 [StrePtomyces coelicolor A3(2)] Val Ile JO-27 NP_631288 secreted Protein Leu Leu Ile Ala Ala Leu Leu Pro 63 [StrePtomyces coelicolor A3(2)] JO-28 NP_630325 secreted Protein Ala Ala Val Val Leu Leu Pro Leu 64 [StrePtomyces coelicolor A3(2)] Ala Ala Ala Pro JO-29 NP_631289 secreted Protein Ala Ala Ala Ala Ala Ala Leu Leu 65 [StrePtomyces coelicolor A3(2)] Val Pro JO-30 CAB51015 Putative secreted Protein Leu Pro Val Val Ala Leu Leu Ala 66 [StrePtomyces coelicolor A3(2)] JO-31 NP_629515 chitinase C (secreted Protein) Ala Ala Ala Leu Ala Ala Pro Leu 67 [StrePtomyces coelicolor A3(2)] Ala Leu Pro JO-32 NP_940995 C1q and tumor necrosis Leu Leu Leu Ala Leu Leu Leu Ala 68 factor related Protein 1 isoform 1 Ala [Homo saPiens] JO-33 NP_854150 POSSIBLE CONSERVED SECRETED Ala Val Ala Val Val Ala Leu Leu 69 PROTEIN [Mycobacterium bovis AF2122/97]

TABLE 1c MTD Origin Amino acid sequence SEQ ID NO JO-34 NP_630361 Probable secreted Protein Leu Leu Leu Ile Ile Val Leu Leu 70 [StrePtomyces coelicolor A3(2)] Ile Val Pro JO-35 P39790 Extracellular metalloProtease Leu Ala Leu Ala Ala Ala Val Val 71 Precursor Pro JO-36 CAA19252 Putative liPoProtein Pro Ala Ala Leu Ala Leu Leu Leu 72 [StrePtomyces coelicolor A3(2)] Val Ala JO-37 P_625685 large secreted Protein Ile Val Ala Leu Leu Leu Val Pro 73 [StrePtomyces coelicolor A3(2)] Leu Val Leu Ala Ile Ala Ala Val Leu JO-38 NP_625685 large secreted Protein Ile Val Ala Leu Leu Leu Val Pro 74 [StrePtomyces coelicolor A3(2)] JO-39 NP_625685 large secreted Protein Pro Leu Val Leu Ala Ile Ala Ala 75 [StrePtomyces coelicolor A3(2)] Val Leu JO-40 NP_808800 golgi PhosPhoProtein 2 Pro Leu Val Leu Ala Ala Leu Val 76 [Homo sapiens] Ala JO-41 NP_626993 selected Protein Ala Ala Ala Leu Leu Ala Val Ala 77 [StrePtomyces coelicolor A3(2)] JO-42 NP_004863 thymic dendritic cell- Pro Leu Leu Leu Leu Ala Leu Ala 78 derived factor 1 [Homo saPiens] JO-43 NP_631398 secreted Protein Ala Leu Ala Leu Val Val Ala 79 [StrePtomyces coelicolor A3(2)] JO-44 NP_627373 Penicillin-binding Protein Val Ala Ala Val Val Val Ala Ala 80 (secreted Protein) [StrePtomyces coelicolor A3(2)] JO-45 NP_056226 sulfatase modifying Pro Leu Leu Pro Leu Leu Leu Leu 81 factor 2 [Homo sapiens] Val JO-46 NP_854998 Conserved hypothelial secreted Val Val Leu Val Val Val Leu Pro 82 protein [Mycobacterium bovis AF2122/97] Leu Ala Val Leu Ala JO-47 NP_627512 secreted Protein Ala Ala Ala Val Pro Val Leu Val 83 [StrePtomyces coelicolor A3(2)] Ala Ala JO-48 NP_110448 phospholipase A2, group Pro Ala Leu Leu Leu Leu Leu Leu 84 XIIA [Homo sapiens] Ala Ala Val Val JO-49 NP_003245 tissue inhibitor of metallo- Pro Leu Ala Ile Leu Leu Leu Leu  85 proteinase 1 precursor [Homo sapiens] Leu Ile Ala Pro JO-50 NP_002978 small inducible cytokine A17 Pro Leu Leu Ala Leu Val Leu Leu 86 precursor [Homo sapiens] Leu Ala Leu Ile Ala JO-51 NP_001012495 stromal cell derived factor Val Val Ala Val Leu Ala Leu Val 87 1 isoform gamma precursor [Mus musculus] Leu Ala Ala Leu

TABLE 1d MTD Origin Amino acid sequence SEQ ID NO J0-52 NP 775628 ficolin 3 isoform 2 Pro Leu Leu Leu Leu Leu Pro Ala 88 precursor [Homo sapiens] Leu JO-53 NP 624483 secreted protein Leu Ala Ala Val Ala Ala Leu Ala 89 [Streptomyces coelicolor A3(2)] Val Val Val Pro JO-54 NP_997465 HERV-FRD provirus Leu Leu Leu Leu Val Leu Ile Leu 90 ancestral Env polyprotein [Homo sapiens] Pro Leu Ala Ala JO-55 NP_854234 posible conserved secreted Leu Ala Val Val Val Val Ala Ala 91 protein [Mycobacterium bovis AF2122/97] Val JO-56 P23284 Peptidyl-prolyl cis-trans Val Leu Leu Ala Ala Ala Leu Ile 92 isomerase B precursor (PPIase) Ala (Rotamase) (Cyclophilin B) JO-57 CAD05047 hypothetical secreted protein Leu Ile Ala Leu Leu Ala Ala Pro 93 [Salmonella enterica subsp. Leu Ala Enterica serovar Typhi] JO-58 P05067 Amyloid beta A4 protein Leu Ala Leu Leu Leu Leu Ala Ala 94 precursor (APP) (ABPP) (Alzheimer disease amyloid protein) JO-59 NP_004878 small inducible cytokine Leu Leu Ala Ala Ala Leu Leu Leu 95 B14 precursor [Homo sapiens] Leu Leu Leu Ala JO-60 NP_626589 secreted protein Val Ile Ile Ala Leu Ile Val Ile 96 [Streptomyces coelicolor A3(2)] Val Ala JO-61 NP_626589 secreted protein Val Val Leu Val Val Ala Ala Val 97 [Streptomyces coelicolor A3(2)] Leu Ala Leu JO-62 NP_856548 SOLUBLE SECRETED ANTIGEN MPB53 Val Ala Val Ala Ile Ala Val Val 98 [Mycobacterium bovis AF2122/97] Leu JO-63 NP_629854 secreted protein Pro Leu Ile Val Val Val Ala Ala 99 [Streptomyces coelicolor A3(2)] Ala Val Val Ala Val JO-64 AAB59058 lambda receptor protein Pro Leu Ala Val Ala Val Ala Ala 100 [Escherichia coli] Val Ala Ala JO-65 NP_825185 NLP/P60-family secreted Ala Ala Ile Ala Leu Val Ala Val 101 protein Val Leu [Streptomyces avermitilis MA-4680] JO-66 NP_626568 secreted protein Ala Ala Ala Leu Ala Ala Ile Ala 102 [Streptomyces coelicolor A3(2)] Val Ile JO-67 NP_626568 secreted protein Ala Ala Ala Pro Ala Val Ala Ala 103 [Streptomyces coelicolor A3(2)]

TABLE 1e MTD Origin Amino acid sequence SEQ ID NO JO-68 NP_625639 secreted protein Leu Leu Leu Ala Ala Leu Pro 104 [Streptomyces coelicolor A3(2)] JO-69 CAC32053 putative secreted protein Ala Leu Leu Ala Val Val Ala Ala 105 [Mycobacterium leprae] JO-70 NP_630954 secreted protein Ala Val Val Val Val Leu Pro Ile 106 [Streptomyces coelicolor A3(2)] Leu Leu JO-71 P97300 Neuroplastin precursor Ala Leu Ala Leu Leu Leu Leu Val 107 (Stromal cell-derived receptor 1) Pro (SDR-1) JO-72 AAA41949 Rat parotid gland acidic Leu Val Val Leu Leu Ala Ala Leu 108 proline-rich protein mRNA, S Leu Val Leu complete CD JO-73 AAA17887 Drosophila melanogaster Pro Val Leu Leu Leu Leu Ala Pro 109 spatzle (spz) gene JO-74 NP_627867 conserved secreted protein Ala Leu Ala Val Val Ala Ala Pro 110 [Streptomyces coelicolor A3(2)] JO-75 NP_631283 secreted protein Val Ile Val Ala Leu Leu Ala Val 111 [Streptomyces coelicolor A3(2)] JO-76 NP_003231 endometrial bleeding  Ala Leu Val Leu Pro Leu Ala Pro   6 associated factor preproprotein [Homo sapiens] JO-77 CAB76313 putative secreted protein Ala Val Ala Leu Leu Ile Leu Ala   8 [Streptomyces coelicolor A3(2)] Val JO-78 P07198 Xenopsin precursor [Contains: Val Leu Leu Ala Val Ile Pro 112 Xenopsin precursor fragment (XPF); Xenopsin] JO-79 NP_631293 secreted protein Leu Ile Val Ala Ala Val Val Val 113 [Streptomyces coelicolor A3(2)] Val Ala Val Leu Ile JO-80 NP_626373 secreted protein Ala Val Val Val Ala Ala Pro 114 [Streptomyces coelicolor A3(2)] JO-81 NP_624952 secreted cellulose-binding Leu Ala Ala Val Leu Leu Leu Ile 115 protein [Streptomyces coelicolor A3(2)] Pro JO-82 NP_009104 protease, serin, 23  Leu Leu Leu Leu Leu Leu Ala Val 116 precursor [Homo sapiens] Val Pro JO-83 AAK63068 phytotoxic protein PcF Ala Val Ala Leu Val Ala Val Val 117 precursor [Phytophthora cactorum] Ala Val Ala JO-84 NC_003903 Streptomyces coelicolor Leu Val Ala Ala Leu Leu Ala Val 118 A3(2) plasmid SCP1, complete sequence Leu

TABLE 1f MTD Origin Amino acid sequence SEQ ID NO JO-85 NP_629842 peptide transport system Leu Leu Ala Ala Ala Ala Ala 119 secreted peptide binding protein Leu Leu Leu Ala [Streptomyces coelicolor A3(2)] JO-86 NP_854067 Posible secreted protein Leu Ala Val Leu Ala Ala Ala Pro 120 [Mycobacterium bovis AF2122/97] JO-87 NP_627802 secreted protein Val Val Val Leu Leu Val Leu Leu 121 [Streptomyces coelicolor A3(2)] Ala Leu Val Val Val JO-88 NP_627802 secreted protein Val Val Ile Ala Val Val Pro 122 [Streptomyces coelicolor A3(2)] JO-89 NP_624483 secreted protein Leu Ala Ala Val Ala Ala Leu Ala 123 [Streptomyces coelicolor A3(2)] Val Val JO-90 NP_627802 secreted protein Val Leu Leu Val Leu Leu Ala Leu 124 [Streptomyces coelicolor A3(2)] Val JO-91 NP_625203 secreted protein Pro Val Leu Val Pro Ala Val Pro 125 [Streptomyces coelicolor A3(2)] JO-92 NP_630960 secreted protein Pro Ala Leu Ala Leu Ala Leu Ala 126 [Streptomyces coelicolor A3(2)] JO-93 NP_630670 secreted protein Ala Ala Ala Ala Pro Ala Leu Ala 127 [Streptomyces coelicolor A3(2)] JO-94 NP_630493 secreted protein Ile Val Leu Pro Val Leu Ala Ala 128 [Streptomyces coelicolor A3(2)] Pro JO-95 CAC29994 putative secreted Leu Val Leu Leu Leu Leu Pro Leu 129 protein [Mycobacterium leprae] Leu Ile JO-96 NP_624483 secreted protein Leu Ala Ala Val Ala Pro Ala Leu 130 [Streptomyces coelicolor A3(2)] Ala Val Val JO-97 NP_037375 secretogranin III Ile Leu Val Leu Val Leu Pro Ile 131 [Homo sapiens] JO-98 NP_009199 V-set and Ile Leu Leu Pro Leu Leu Leu Leu 132 immunoglobulin domain containing Pro 4 [Homo sapiens] JO-99 NP_733650 secreted hydrolase Ile Ala Pro Ala Val Val Ala Ala 133 [Streptomyces coelicolor A3(2)] Leu Pro JO-100 NP_057540 transmembrane protein 9 Leu Leu Leu Val Ala Val Val Pro 134 [Homo sapiens] Leu Leu Val Pro JO-76 CAI74362 hypothetical protein Leu Ile Leu Leu Leu Leu Pro Ile 135 [Theileria annulata] Ile JO-102 NP-630671 secreted protein Ala Val Leu Ala Ala Pro Ala Val 136 [Streptomyces coelicolor A3(2)] Leu Val JO-77 NP_065695 TMEM9 domain family, Leu Ala Leu Pro Val Leu Leu Leu 137 member B [Homo sapiens] Ala JO-104 P06908 Pulmonary surfactant- Leu Ala Leu Ala Leu Leu Leu 138 associated protein A precursor (SP-A) (PSP-A) (PSAP)

TABLE 1g MTD Origin Amino acid sequence SEQ ID NO JO-105 NP_639721 putative secreted protein Val Ala Val Pro Leu Leu Val Val 139 [Streptomyces coelicolor A3(2)] Ala JO-106 NP_854954 CONSERVED PROBABLE SECRETED Ala Val Ala Val Ala Pro Val Ala 140 PROTEIN [Mycobacterium bovis AF2122/97] Ala Ala Ala JO-107 NP_627759 secreted protein Ala Ala Ala Val Val Ala Ala Val 141 [Streptomyces coelicolor A3(2)] Pro Ala Ala JO-108 NP_003842 cellular repressor of Ala Leu Leu Ala Ala Leu Leu Ala 142 E1A-stimulated genes [Homo sapiens] Pro JO-109 NP_003842 cellular repressor of Leu Leu Ala Leu Leu Val Pro 143 E1A-stimulated genes [Homo sapiens] JO-110 NP_003842 cellular repressor of Ala Leu Leu Ala Ala Leu Leu Ala 144 E1A-stimulated genes [Homo sapiens] Leu Leu Ala Leu Leu Val JO-111 NP_000589 Homo sapiens insulin-like Ala Ala Ala Leu Pro Leu Leu Val 145 growth factor binding protein 3 (IGFBP3) Leu Leu Pro JO-112 CAB594459 putative secreted protein Ala Ala Ala Val Pro Ala Ala Leu 146 [Streptomyces coelicolor A3(2)] Ala Pro JO-113 NP_628917 secreted protein Ala Ala Leu Ala Val Ala Ala Leu 147 [Streptomyces coelicolor A3(2)] Ala Ala JO-114 NP_624695 secreted protein Ala Val Leu Ala Ala Ala Val Pro 148 [Streptomyces coelicolor A3(2)] JO-115 NP_624695 secreted protein Val Ala Ala Leu Pro Ala Pro Ala 149 [Streptomyces coelicolor A3(2)] JO-116 NP_624791 secreted protein Ala Leu Ala Leu Ala Val Pro Ala 150 [Streptomyces coelicolor A3(2)] Val Leu Pro JO-117 CAB45579 putative secreted protein Ala Ala Leu Leu Pro Ala Ala Val 151 [Streptomyces coelicolor A3(2)] Ala Val Pro JO-118 NP_627066 secreted protein Ala Val Val Val Ala Leu Ala Pro 152 [Streptomyces coelicolor A3(2)] JO-119 NP_630174 secreted substrate-binding Ala Ala Ala Val Ala Leu Pro Ala 153 protein [Streptomyces coelicolor A3(2)] Ala Ala Ala Leu Leu Ala JO-120 P06727 Apolipoprotein A-IV precursor Ala Val Val Leu Pro Leu Ala Leu 154 (Apo-AIV) (ApoA-IV) Homo sapiens Val Ala Val Ala Pro JO-121 Q62087 Serum paraoxonase/lactonase 3. Leu Val Ala Leu Pro Leu Leu Pro 155 Mus musculus JO-122 NP_627123 probable secreted penicillin- Val Val Val Pro Leu Leu Leu Ile 156 binding protein Val Pro [Streptomyces coelicolor A3(2)]

TABLE 1h MTD Origin Amino acid sequence SEQ ID NO JO-123 CAC30224 putative secreted  Leu Ala Val Val Leu Ala Val 157 hydrolase [Mycobacterium leprae] Pro JO-124 OZZQAM circumsporozoite protein Leu Leu Ala Val Pro Ile Leu 158 precursor-Plasmodium cynomolgi Leu Val Pro JO-125 Q15166 Serum paraoxonase/lactonase Leu Val Ala Leu Val Leu Leu 159 3 [Homo sapiens] Pro JO-126 NP_060220 all-trans-13,14- Leu Val Leu Leu Leu Ala Val 160 dihydroretinol saturase [Homo sapiens] Leu Leu Leu Ala Val Leu Pro JO-127 AL627273 Salmonella enterica serovar Leu Leu Ala Pro Val Val Ala 161 Typhi (Salmonella typhi) strain CT18 Leu Val Ile Leu Pro JO-128 NP_625987 secreted protein Val Leu Ala Val Leu Ala Val 162 [Streptomyces coelicolor A3(2)] Pro Val Leu Leu Leu Pro JO-129 CAB45474 putative secreted protein Val Val Ile Ala Val Val Pro 163 [Streptomyces coelicolor A3(2)] Val Val Val JO-130 CAB45474 putative secreted protein Leu Leu Val Leu Leu Ala Leu 164 [Streptomyces coelicolor A3(2)] Val Val Val Pro JO-131 CAB36605 putative secreted protein Val Leu Leu Ala Leu Pro Val 165 [Streptomyces coelicolor A3(2)] Val Ala Ala Pro JO-132 NP_628377NLP/P60-family secreted Ala Val Val Val Pro Ala Ile 166 protein [Streptomyces coelicolor A3(2)] Val Leu Ala Ala Pro JO-133 CAB59594 putative secreted protein Ala Val Leu Val Pro Ala Ala 167 [Streptomyces coelicolor A3(2)] Ala Leu Val Pro JO-134 NP_624974 secreted protein Val Val Ala Ala Leu Pro Leu 168 [Streptomyces coelicolor A3(2)] Val Leu Pro JO-135 NP_733682 secreted ATP/GTP binding Ala Ala Val Ala Leu Pro Ala 169 protein [Streptomyces coelicolor A3(2)] Ala Ala Pro JO-136 P27169 Serum paraoxonase/arylesterase 1 Leu Ile Ala Leu Pro Leu Leu 170 (PON 1) (Serum aryldialkylphosphatase 1) Pro (A-esterase 1) Homo sapiens JO-137 P52430 Serum paraoxonase/arylesterase 1 Leu Leu Ala Leu Pro Leu Val 171 (PON 1) (Serum aryldialkylphosphatase 1) Leu Val Leu Ala Leu Pro (A-esterase 1) Homo sapiens

TABLE 1i MTD Origin Amino acid sequence SEQ ID NO JO-138 NP_626569 secreted protein Ile Val Pro Leu Leu Leu Ala Ala 172 [Streptomyces coelicolor A3(2)] Pro JO-139 NP_940995 C1q and tumor necrosis Leu Leu Leu Ala Pro Leu Leu Leu 173 factor related protein 1 isoform 1 Ala Pro [Homo sapiens] JO-140 NP_626174 large secreted protein Leu Ala Ala Leu Pro Val Ala Ala 174 [Streptomyces coelicolor A3(2)] Val Pro JO-141 CAB83860 putative protein-export Ala Leu Ala Val Ile Val Leu Val 175 integral membrane protein  Leu Leu [Neisseria meningitidis Z2491] JO-142 NP_001009551 cornichon-like Leu Ala Leu Leu Leu Pro Ala Ala 176 isoform 2 [Homo sapiens] Leu Ile JO-143 NP_626808 secreted protein Ala Leu Leu Pro Leu Leu Ala Val 177 [Streptomyces coelicolor A3(2)] Val Leu Pro JO-144 NP_639798 putative secreted protein Ala Ile Ala Val Pro Val Leu Ala 178 [Streptomyces coelicolor A3(2)] Ala Pro JO-145 NP_000492 Homo sapiens elastin Ala Ala Ala Pro Val Leu Leu Leu 179 (supravalvular aortic stenosis) Leu Leu JO-146 NP_630680 secreted sugar binding Ala Ala Ala Val Ala Val Leu Ala 180 protein [Streptomyces coelicolor A3(2)] Leu Ala Pro JO-147 CAB56129 putative secreted protein Ala Ala Leu Ala Ala Leu Val Val 181 [Streptomyces coelicolor A3(2)] Ala Ala Pro JO-148 NP_625109 secreted solute-binding lipo- Ala Ala Leu Ala Ala Val Pro Leu 182 protein [Streptomyces coelicolor A3(2)] Ala Leu Ala Pro JO-149 NP_733579 secreted sugar-binding Ala Leu Ala Val Ala Ala Pro Ala 183 protein [Streptomyces coelicolor A3(2)] Leu Ala Leu Leu Pro JO-150 NP_630126 secreted chitinase Ala Ala Leu Pro Ala Ala Ala Pro 184 (secreted protein) [Streptomyces coelicolor A3(2)] JO-151 NP_630126 secreted chitinase Ala Ala Ala Pro Val Ala Ala Val 185 (secreted protein) Pro [Streptomyces coelicolor A3(2)] JO-152 NP_872425 secretory protein Leu Leu Ala Val Leu Leu Ala Leu 186 LOC348174 [Homo sapiens] Leu Pro JO-153 NP_630107 secreted protein Val Leu Ala Leu Leu Val Ala Val 187 [Streptomyces coelicolor A3(2)] Val Pro

TABLE 1j MTD Origin Amino acid sequence SEQ ID NO JO-154 NP_733688 peptide-binding transport Ala Leu Val Val Pro Ala Ala Val 188 protein [Streptomyces coelicolor A3(2)] Pro JO-155 NP_629904 secreted protein Ala Val Val Leu Pro Leu Leu Leu 189 [Streptomyces coelicolor A3(2)] Pro JO-156 YP_177852 MCE-FAMILY PROTEIN MCE3A Ala Val Ile Pro Val Ala Val Leu 190 [Mycobacterium tuberculosis H37Rv] Val Pro JO-157 CAA19627 putative secreted solute Ala Ala Ala Val Pro Ala Ala Val 191 binding protein Leu Ala Pro [Streptomyces coelicolor A3(2)] JO-158 NP_639884 putative large secreted Val Ala Val Pro Val Val Leu Ala 192 protein [Streptomyces coelicolor A3(2)] Ile Leu Pro JO-159 P24327 Foldase protein prsA precursor Ile Ala Ile Ala Ala Ile Pro Ala 193 Ile Leu Ala Leu JO-160 CAB84808 putative membrane lipoprotein Ala Leu Ile Ala Pro Ala Leu Ala 194 [Neisseria meningitidis Z2491] Ala Pro JO-161 NP_639883 putative large secreted Ala Ala Ile Ala Leu Val Ala Pro 195 protein [Streptomyces coelicolor A3(2)] Ala Leu JO-162 NP_639883 putative large secreted Leu Ala Pro Ala Val Ala Ala Ala 196 protein [Streptomyces coelicolor A3(2)] Pro JO-163 NP_627362 secreted protein Val Ala Ile Ile Val Pro Ala Val 197 [Streptomyces coelicolor A3(2)] Val Ala Ile Ala Leu Ile Ile JO-164 NP_627362 secreted protein Ala Val Val Ala Ile Ala Leu Ile 198 [Streptomyces coelicolor A3(2)] Ile JO-165 NP_624625 secreted protein Leu Ala Ala Val Pro Ala Ala Ala 199 [Streptomyces coelicolor A3(2)] Pro JO-166 NP_624625 secreted protein Ala Val Ala Ala Leu Pro Leu Ala 200 [Streptomyces coelicolor A3(2)] Ala Pro JO-167 NP_624625 secreted protein Leu Ala Ala Pro Ala Ala Ala Ala 201 [Streptomyces coelicolor A3(2)] Pro JO-168 NP_626936 secreted protein Leu Ala Ala Val Val Pro Val Ala 202 [Streptomyces coelicolor A3(2)] Ala Ala Val Pro JO-169 NP_626936 secreted protein Val Ala Ala Pro Ala Ala Ala Ala 203 [Streptomyces coelicolor A3(2)] Pro JO-170 NP_626936 secreted protein Ala Val Pro Val Pro Val Pro Leu 204 [Streptomyces coelicolor A3(2)]

TABLE 1k MTD Origin Amino acid sequence SEQ ID NO JO-171 NP_085072 matrilin 2 isoform b Leu Leu Ile Leu Pro Ile Val Leu 205 precursor [Homo sapiens] Leu Pro JO-172 CAB94057 putative secreted protein Ala Leu Ala Leu Pro Ala Leu Ala 206 [Streptomyces coelicolor A3(2)] Ile Ala Pro JO-173 NP_624384 secreted protein Ala Val Ile Pro Ile Leu Ala Val 207 [Streptomyces coelicolor A3(2)] Pro JO-174 NP_733505 large, multifunctional Leu Ile Leu Leu Leu Pro Ala Val 208 secreted protein Ala Leu Pro [Streptomyces coelicolor A3(2)] JO-175 CAB45630 putative secreted protein Ile Val Leu Ala Pro Val Pro Ala 209 [Streptomyces coelicolor A3(2)] Ala Ala JO-176 NP_627887 secreted protein Val Val Val Val Pro Val Leu Ala 210 [Streptomyces coelicolor A3(2)] Ala Ala Ala JO-177 P06832 Bacillolysin precursor Leu Val Ala Val Ala Ala Pro 211 JO-178 NP_625998 secreted protein Leu Val Leu Ala Ala Pro Ala Ala 212 [Streptomyces coelicolor A3(2)] Leu Pro JO-179 NP_625057 secreted protein Leu Ile Ala Pro Ala Ala Ala Val 213 [Streptomyces coelicolor A3(2)] Pro JO-180 NP_443750 ADP-ribosyltransferase Ala Leu Ala Ala Leu Pro Ile Ala 214 5 precursor [Homo sapiens] Leu Pro JO-181 CAB84257 putative secreted protein Ala Val Leu Leu Leu Pro Ala Ala 215 [Neisseria meningitidis Z2491] Ala JO-182 P00634 Alkaline phosphatase Ile Ala Leu Ala Leu Leu Pro Leu 216 precursor (APase) Leu JO-183 NP_000933 peptidylprolyl isomerase Val Leu Leu Ala Ala Ala Leu Ile 217 B precursor [Homo sapiens] Ala Pro JO-184 CAB71258 putative secreted protein Ala Pro Ala Val Leu Pro Pro Val 218 [Streptomyces coelicolor A3(2)] Val Val Ile JO-185 CAC31847 possible secreted protein Val Val Gly Leu Leu Val Ala Ala 219 [Mycobacterium leprae] Leu JO-186 NP_626948 secreted protein Ala Ala Ile Ala Ala Ala Ala Pro 220 [Streptomyces coelicolor A3(2)] Leu Ala Ala JO-187 NP_059120 cat eye syndrome critical Leu Leu Leu Ala Val Ala Pro 221 region protein 1 isoform a  precursor [Homo sapiens] JO-188 NP_006519 tissue factor pathway Leu Ile Leu Leu Leu Pro Leu Ala 222 inhibitor 2 [Homo sapiens] Ala Leu

TABLE 1l MTD Origin Amino acid sequence SEQ ID NO JO-189 P97299 Secreted frizzled-relating Ala Leu Leu Leu Leu Val Leu 223 protein 2 precursor (sFRP-2) Ala (Secreted apoptosis-relating protein 1) JO-190 NP_071447 tubulointerstitial Leu Leu Leu Leu Leu Leu Pro 224 nephritis antigen-like 1 Leu Ala JO-191 NP_056322 epidermal growth factor-like Leu Ala Leu Pro Leu Leu Leu 225 protein 6 precursor [Homo sapiens] Pro JO-192 NP_628035 secreted penicillin-binding Leu Leu Val Leu Pro Leu Leu 226 protein [Streptomyces coelicolor A3(2)] Ile JO-193 NP_683880 cathepsin H isoform b Leu Pro Leu Leu Pro Ala Ala 227 precursor [Homo sapiens] Leu Val kFGF4- kaposi fibroblast growth factor 4, Ala Ala Val Leu Leu Pro Val 228 derived kFGF4 Leu Leu Ala Ala Pro MTD

In some embodiments, the present invention may employ a kaposi fibroblast growth factor 4 (kFGF4)-derived MTD having the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4 (hereinafter, “MTD₁”), a JO-76 MTD having the nucleotide sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6 which is a hypothetical protein derived from Theileria annulata (hereinafter, “MTD₂”), and a JO-77 MTD having the nucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8 which belongs to member B of a human TMEM9 domain family (hereinafter, “MTD₃”), as the MTD capable of mediating the transport of the metastasis suppressor Nm23 into a cell.

The cell permeable Nm23 recombinant proteins according to the present invention have a structure where one of the three MTDs (kFGF4-derived MTD: MTD₁, JO-76: MTD₂, JO-77: MTD₃) is fused to one terminus or both termini of a metastasis suppressor protein Nm23, and a SV40 large T antigen-derived nuclear localization sequence (NLS)(nucleotide sequence of SEQ ID NO: 9, amino acid sequence of SEQ ID NO:10) and a histidine-tag (His-Tag) affinity domain for easy purification are fused to one terminus of the resulting construct.

In another embodiment, the present invention relates to the construction of eight full-length forms of a cell permeable Nm23 recombinant protein by using one of a kFGF4-derived MTD, a JO-76 MTD and a JO-77 MTD.

As used herein, the term “full-length form” refers to a construct including the entire amino acid sequence of the metastasis suppressor protein Nm23 which does not contain any deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2. However, it should be obvious to a skilled person in the art that Nm23 derivatives including various kinds of modifications through the deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2 that is made within the scope of not causing a deterioration of the Nm23 anti-metastatic effect can be used in the present invention.

Referring to FIG. 1, the full-length forms of the cell permeable Nm23 recombinant protein are as follows:

1) His-MTD₁-Nm23 (HM₁N), where a kFGF4-derived MTD is fused to the N-terminus of a full-length Nm23,

2) His-Nm23-MTD₁ (HNM₁), where a kFGF4-derived MTD is fused to the C-terminus of a full-length Nm23,

3) His-MTD₁-Nm23-MTD₁ (HM₁NM₁) where a kFGF4-derived MTD is fused to both termini of a full-length Nm23,

4) His-MTD₂-Nm23 (HM₂N), where a JO-76 MTD is fused to the N-terminus of a full-length Nm23,

5) His-Nm23-MTD₂ (HNM₂), where a JO-76 MTD is fused to the C-terminus of a full-length Nm23,

6) His-MTD₃-Nm23 (HM₃N), where a JO-77 MTD is fused to the N-terminus of a full-length Nm23,

7) His-Nm23-MTD₃ (HNM₃), where a JO-77 MTD is fused to the C-terminus of a full-length Nm23, and

8) His-MTD₃-Nm23-MTD₃ (HM₃NM₃), where a JO-77 MTD is fused to both termini of a full-length Nm23,

where a His-tag and a NLS derived from SV40 large T antigen are covalently coupled to the N-terminus of the above constructs.

As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a kFGF4-derived MTD as described above, His-MTD₁-Nm23 (HM₁N) has an amino acid sequence represented by SEQ ID NO: 22, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 21; His-Nm23-MTD₁ (HNM₁) has an amino acid sequence represented by SEQ ID NO: 24, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 23; and His-MTD₁-Nm23-MTD₁ (HM₁NM₁) has an amino acid sequence represented by SEQ ID NO: 26, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 25.

As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a JO-76 MTD as described above, His-MTD₂-Nm23 (HM₂N) has an amino acid sequence represented by SEQ ID NO: 28, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 27; and His-Nm23-MTD₂ (HNM₂) has an amino acid sequence represented by SEQ ID NO: 30, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 29.

As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a JO-77 MTD as described above, His-MTD₃-Nm23 (HM₃N) has an amino acid sequence represented by SEQ ID NO: 32, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 31; His-Nm23-MTD₃ (HNM₃) has an amino acid sequence represented by SEQ ID NO: 34, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 33; and His-MTD₃-Nm23-MTD₃ (HM₃NM₃) has an amino acid sequence represented by SEQ ID NO: 36, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 35.

As a control for the cell permeable Nm23 recombinant proteins, His-Nm23 (HN), where a full-length Nm23 is fused only to a NLS derived from SV40 large T antigen and a histidine-tag (His-Tag) without any MTD, is constructed. The control protein has an amino acid sequence represented by SEQ ID NO: 20, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 19.

Further, the present invention provides an expression vector containing the polynucleotide encoding each of the cell permeable Nm23 recombinant proteins described above, and a transformant capable of producing each of the cell permeable Nm23 recombinant proteins at high levels, which is obtainable by transforming a host cell using the expression vector.

As used herein, the term “expression vector” is a vector capable of expressing a target protein or a target RNA in a suitable host cell. The nucleotide sequence of the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host cell.

Within an expression vector, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence. The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements. Such operable linkage with the expression vector can be achieved by conventional gene recombination techniques known in the art, while site-directed DNA cleavage and linkage are carried out by using conventional enzymes known in the art.

The expression vectors suitable for the present invention may include plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors and the like, but are not limited thereto. The expression vectors for use in the present invention may contain a signal sequence or a leader sequence for membrane targeting or secretion, as well as regulatory sequences such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer and the like. The promoter may be a constitutive or an inducible promoter. Further, the expression vector may include one or more selectable marker genes for selecting the host cell containing the expression vector, and may further include a nucleotide sequence that enables the vector to replicate in the host cell in question.

The expression vector constructed according to the present invention may be exemplified by pET28a(+)-HNM₁ where the polynucleotide encoding the recombinant protein HNM₁ where a kFGF4-derived MTD is fused to the C-terminus of a full-length Nm23 is inserted into a cleavage site of NdeI restriction enzyme within the multiple cloning sites (MCS) of a pET-28a(+) vector.

In another embodiment, the polynucleotide of the present invention is cloned into a pET-28a(+) vector (NOVAGEN, USA) bearing a His-tag sequence so as to fuse six histidine residues to the N-terminus of the cell permeable Nm23 recombinant protein to allow easy purification.

Accordingly, the cell permeable Nm23 recombinant protein expressed in the above expression vector has a structure where one of a kFGF4-derived MTD, a JO-76 MTD and a JO-77 MTD is fused to the full-length or truncated Nm23, and a His-tag and NLS are linked to the N-terminus thereof.

The present invention further provides a transformant capable of producing each of the cell permeable Nm23 recombinant proteins at high levels which is obtainable by transforming a host cell using the expression vector. The host cell suitable for the present invention may be eukaryotic cells, such as E. coli. In one embodiment of the present invention, E. coli used as a host cell is transformed with the expression vector, for example, pET28a(+)-HNM₁ containing the polynucleotide encoding the cell permeable recombinant protein HNM₁ where a kFGF4-derived MTD is fused to the C-terminus of a full-length Nm23 according to the present invention so as to produce the cell permeable Nm23 recombinant protein at high levels. Methods for transforming bacterial cells are well known in the art, and include, but are not limited to, biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, microprojectile bombardment, calcium phosphate (CaPO₄) precipitation, calcium chloride (CaCl₂) precipitation, PEG-mediated fusion and liposome-mediated method.

In some embodiments, the transformants obtained by transforming E. coli DH5α with the expression vector containing the cell permeable Nm23 recombinant protein HM₃N where a JO-77 MTD is fused to the N-terminus of a full-length Nm23, and the expression vector containing the cell permeable Nm23 recombinant protein HNM₃ where a JO-77 MTD is fused to the C-terminus thereof, respectively, were deposited under accession numbers KCTC-11380BP and KCTC-11381BP, respectively, with the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong, Yusong-Ku, Taejon 305-333, Republic of Korea. All deposits referred to herein were made on Aug. 28, 2008 in accordance with the Budapest Treaty, and all restrictions imposed by the depositor on the availability to the public of the deposited biological material will be irrevocably removed upon the granting of the patent.

The present invention provides a method of producing the cell permeable Nm23 recombinant proteins at high levels, which includes the step of culturing the above transformant.

The method of the present invention may be carried out by culturing the transformant in a suitable medium under suitable conditions for expressing a cell permeable Nm23 recombinant protein of the present invention in the expression vector introduced into the transformant. Methods for expressing a recombinant protein by culturing a transformant are well known in the art, and for example, may be carried out by inoculating a transformant in a suitable medium for growing the transformant, performing a subculture, transferring the same to a main culture medium, culturing under suitable conditions, for example, supplemented with a gene expression inducer, isopropyl-J3-D-thiogalactoside (IPTG) and, thereby, inducing the expression of a recombinant protein. After the culture is completed, it is possible to recover a “substantially pure” recombinant protein from the culture solution. The term “substantially pure” means that the recombinant protein and polynucleotide encoding the same of the present invention are essentially free of other substances with which they may be found in nature or in vivo systems to the extent practical and appropriate for their intended use.

A recombinant protein of the present invention obtained as above may be isolated from the inside or outside (e.g., medium) of host cells, and purified as a substantially pure homogeneous polypeptide. The method for polypeptide isolation and purification is not limited to any specific method. In fact, any standard method may be used. For instance, chromatography, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization may be appropriately selected and combined to isolate and purify the polypeptide. As for chromatography, affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography, etc., for example, may be used (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989; Deutscher, M., Guide to Protein Purification Methods Enzymology vol. 182. Academic Press. Inc., San Diego, Calif., 1990).

Meanwhile, the recombinant protein expressed in the transformants according to the present invention can be classified into a soluble fraction and an insoluble fraction according to protein characteristics during the protein purification process. If the majority of the expressed recombinant proteins are present in the soluble fraction, the recombinant protein can be isolated and purified according to the method as described above. However, when the majority of the expressed recombinant proteins are present in the insoluble fraction, i.e., as inclusion bodies, the recombinant proteins are first solubilized by using polypeptide denaturing agents, e.g., urea, guanidine HCl, or detergents, and then, purified by performing a series of centrifugation, dialysis, electrophoresis and column chromatography. Since there is the risk of losing the recombinant protein's activity due to a structural modification caused by the polypeptide denaturing agent, the process of purifying the recombinant protein from the insoluble fraction requires desalting and refolding steps. That is, the desalting and refolding steps can be performed by dialysis and dilution with a solution that does not include a polypeptide denaturing agent or by centrifugation with a filter. Further, if a salt concentration of the solution used for the purification of a recombinant protein from a soluble fraction is relatively high, such desalting and refolding steps may be performed.

In some embodiments, it has been found that the cell permeable Nm23 recombinant protein of the present invention mostly exists in the insoluble fraction as an inclusion body. In order to purify the recombinant protein from the insoluble fraction, the insoluble fraction may be dissolved in a lysis buffer containing a non-ionic surfactant such as Triton X-100, subjected to ultrasonification, and then centrifuged to separate a precipitate. The separated precipitate may be dissolved in a buffer supplemented with a strong denaturing agent, such as urea, and centrifuged to separate the supernatant. The above separated supernatant is purified by means of a histidin-tagged protein purification kit and subjected to ultrafiltration, for example, by using an amicon filter for salt removal and protein refolding, thereby obtaining a purified recombinant protein of the present invention.

Further, the present invention provides an anti-metastatic pharmaceutical composition including the cell permeable Nm23 recombinant protein as an effective ingredient for preventing metastasis by inhibiting the proliferation, differentiation and migration of cancer cells and inducing apoptosis.

The metastasis suppressor Nm23, which functions as an important target protein for signal transduction cascades including KSR and Ras-mediated MAPK, can control the MAPK signal transduction cascade mediated by KSR phosphorylation, and thus, inhibit the proliferation, differentiation and migration of cancer cells and induce apoptosis. Therefore, the cell permeable Nm23 recombinant proteins of the present invention can be effectively used as an anti-metastatic agent capable of preventing and/or treating cancer metastasis.

The cell permeable Nm23 recombinant proteins of the present invention can activate cell signaling mechanisms involved in the activation of ATM and p53 that induce cell cycle arrest and apoptosis in response to DNA damage or oncogenic signals by efficiently introducing a metastasis suppressor protein Nm23 into a cell. Therefore, the cell permeable Nm23 recombinant proteins of the present invention can be effectively used as an anti-metastatic agent for treating various kinds of human cancers.

The pharmaceutical composition comprising the recombinant protein of the present invention as an effective ingredient may further include pharmaceutically acceptable carriers suitable for oral administration or parenteral administration. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995). The carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. In case of oral administration, the recombinant protein of the present invention can be formulated in the form of chewable tablets, buccal tablets, troches, capsules, elixir, suspensions, syrup, wafers or combination thereof by mixing with the carriers. Further, the carriers for parenteral administration may include water, suitable oil, saline, aqueous glucose, glycol and the like, and may further include stabilizers and preservatives. The stabilizers suitable for the present invention may include antioxidants such as sodium bisulfite, sodium sulfite and ascorbic acid. Suitable preservatives may include benzalconium chloride, methly-paraben, propyl-paraben and chlorobutanol.

The pharmaceutical composition of the present invention may be formulated into various parenteral or oral administration forms. Representative examples of the parenteral formulation include those designed for administration by injection. For injection, the recombinant proteins of the present invention may be formulated in aqueous solutions, specifically in physiologically compatible buffers or physiological saline buffer. These injection formulations may be formulated by conventional methods using one or more dispersing agents, wetting agents and suspending agents. For oral administration, the proteins can be readily formulated by combining the proteins with pharmaceutically acceptable carriers well known in the art. Such carriers enable the proteins of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Such oral solid formulations may include suitable excipients such as diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol cellulose and/or glycin) and lubricants (e.g., colloidal silica, talc, stearic acid, magnesium stearate, calcium stearate, and/or polyethylene glycol). The tablets may include binders, such as aluminum silicate, starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP), and disintegrating agents, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, absorbents, coloring agents, flavoring agents and/or sweeteners may be added. The formulations can be prepared by mixing, granulating or coating according to conventional methods well-known in the art.

If necessary, the pharmaceutical compositions of the present invention may further include pharmaceutical additives, such as preservatives, antioxidants, emulsifiers, buffering agents and/or salts for regulating osmosis and other therapeutically effective materials, and can be formulated according to conventional methods known in the art.

In addition, the pharmaceutical composition of the present invention can be administered via oral routes or parenteral routes such as intravenously, subcutaneously, intranasally or intraperitoneally. The oral administration may include sublingual application. The parenteral administration may include drip infusion and injection such as subcutaneous injection, intramuscular injection, intravenous injection and introtumoral injection.

The total effective amount of the recombinant protein of the present invention can be administered to patients in a single dose or can be administered by a fractionated treatment protocol, in which multiple doses are administered over a more prolonged period of time. Although the amount of the recombinant protein or nucleic acid encoding the same in the pharmaceutical composition of the present invention may vary depending on the severity of diseases, the protein or the nucleic acid may be generally administered several times a day at an effective dose of 5 to 20 mg. However, a suitable dose of the recombinant protein in the pharmaceutical composition of the present invention may depend on many factors, such as age, body weight, health condition, sex, disease severity, diet and excretion of patients, as well as the route of administration and the number of treatments to be administered. In view of the above factors, any person skilled in the art may determine the effective dose of the recombinant protein as an anti-metastatic agent for preventing metastasis in various human cancers. The pharmaceutical composition of the present invention containing the recombinant protein has no special limitations on its formulation, administration route and/or administration mode insofar as it exhibits the effects of the present invention.

EXAMPLES

The following examples are provided to illustrate the embodiments of the present invention in more detail, but are by no means intended to limit its scope.

Example 1 Construction of Cell Permeable Nm23 Recombinant Proteins (CP-Nm23)

Eight full-length forms of a cell permeable Nm23 (CP-Nm23) recombinant protein were constructed by using one of a kFGF4-derived MTD (MTD₁), a JO-76MTD (MTD₃) and a JO-77 MTD (MTD₂) as a macromolecule transduction domain.

Referring to FIG. 1, the full-length forms of CP-Nm23 recombinant constructs were as follows:

1) His-MTD₁-Nm23 (HM₁N), where a kFGF4-derived MTD is fused to the N-terminus of a full-length Nm23,

2) His-Nm23-MTD₁ (HNM₁), where a kFGF4-derived MTD is fused to the C-terminus of a full-length Nm23,

3) His-MTD₁-Nm23-MTD₁ (HM₁NM₁) where a kFGF4-derived MTD is fused to both termini of a full-length Nm23,

4) His-MTD₂-Nm23 (HM₂N), where a JO-76 MTD is fused to the N-terminus of a full-length Nm23,

5) His-Nm23-MTD₂ (HNM₂), where a JO-76 MTD is fused to the C-terminus of a full-length Nm23,

6) His-MTD₃-Nm23 (HM₃N), where a JO-77 MTD is fused to the N-terminus of a full-length Nm23,

7) His-Nm23-MTD₃ (HNM₃), where a JO-77 MTD is fused to the C-terminus of a full-length Nm23, and

8) His-MTD₃-Nm23-MTD₃ (HM₃NM₃), where a JO-77 MTD is fused to both termini of a full-length Nm23,

where a His-tag and a NLS derived from SV40 large T antigen are covalently coupled to the N-terminus of the above constructs.

In order to prepare the full-length CP-Nm23 recombinant constructs, polymerase chain reactions (PCRs) were carried out by using the oligonucleotides described in Table 1 below as a primer pair specific for each recombinant construct and a human Nm23 cDNA (SEQ ID NO: 1) as a template. The forward and reverse primers for amplifying HM₁N have nucleotide sequences represented by SEQ ID NOS: 13 and 12, respectively; those for amplifying HNM₁ have nucleotide sequences represented by SEQ ID NOS: 11 and 14, respectively; those for amplifying HM₁NM₁ have nucleotide sequences represented by SEQ ID NOS: 13 and 14, respectively; those for amplifying HM₂N have nucleotide sequences represented by SEQ ID NOS: 15 and 12, respectively; those for amplifying HNM₂ have nucleotide sequences represented by SEQ ID NOS: 11 and 16, respectively; those for amplifying HM₃N have nucleotide sequences represented by SEQ ID NOS: 17 and 12, respectively; those for amplifying HNM₃ have nucleotide sequences represented by SEQ ID NOS: 11 and 18, respectively; and those for amplifying HM₃NM₃ have nucleotide sequences represented by SEQ ID NOS: 17 and 18, respectively.

The oligonucleotides as a forward and reverse primer set specific for each recombinant protein are summarized in Table 2 below.

TABLE 2 SEQ Primer ID NO Sequence HN-5′ 11 CCG CAT ATG GCC AAC TGT GAG CGT ACC (30 nts) TCC HN-3′ 12 CCG CAT ATG TCA TTC ATA GAT CCA GTT (33 nts) CTG AGC HM₁N-5′ 13 CCG CAT ATG GCA GCC GTT CTT CTC CCT (72 nts) GTT CTT CTT GCC GCA CCC GCC AAC TGT GAG CGT ACC TTC ATT GCG HNM₁-3′ 14 CCG CAT ATG TCA GGG TGC GGC AAG AAG (72 nts) AAC AGG GAG AAG AAC GGC TGC TTC ATA GAT CCA GTT CTG AGC ACA HM₂N-5′ 15 CCG CAT ATG GCG CTG GTG CTG CCG CTG (60 nts) GCG CCG GCC AAC TGT GAG CGT ACC TTC ATT GCG HNM₂-3′ 16 CCG CAT ATG TCA CGG CGC CAG CGG CAG (60 nts) CAC CAG CGC TTC ATA GAT CCA GTT CTG AGC ACA HM₃N-5′ 17 CCG CAT ATG GCG GTG GCG CTG CTG ATT (63 nts) CTG GCG GTG GCC AAC TGT GAG CGT ACC TTC ATT GCG HNM₃-3′ 18 CCG CAT ATG TCA CAC CGC CAG AAT CAG (63 nts) CAG CGC CAC CGC TTC ATA GAT CCA GTT CTG AGC ACA

The PCR was performed in a 50 μl reaction mixture containing 100 ng of human Nm23 cDNA as a template, 0.2 mM dNTP mixture (dGTP, dATP, dTTP, and dCTP, each at 2 mM), 0.6 μM of each primer, 5 of 10×Taq buffer, 1 μl of Taq polymerase (TAKARA, Japan). The PCR was performed for 25 cycles at 94° C. for 45 seconds, at 53° C. for 45 seconds and at 72° C. for 45 seconds after the initial denaturation of 94° C. for 2 minutes, followed by the final extension at 72° C. for 5 minutes. After the PCR was completed, the amplified PCR product was digested with restriction enzyme Ndel and loaded onto a 1.0% agarose gel and fractionated. As shown in FIGS. 2 a and 2 b, it was confirmed that the expected fragment for each recombinant construct fused to one of a kFGF4-derived MTD, a JO-76 MTD and a JO-77 MTD was successfully amplified.

The DNA band of expected size was excised from the gel, eluted, and purified by using a QIAquick™ Gel extraction kit (QIAGEN, USA). The eluted DNA was precipitated with ethanol and resuspended in distilled water for ligation. As shown in FIG. 3 a, the PCR amplified DNA fragment containing the coding region was subcloned into a pGEM-T Easy vector (PROMEGA, Madison Wis. USA) with a T4 ligase according to the TA cloning method, and then, followed by transformation of E. coli DH5α competent cells with the pGEM-T Easy vector. The cells were plated onto LB plate media supplemented with 50 μg/ml of ampicillin and cultured at 37° C. for overnight. After the recombinant fragment-inserted pGEM-T Easy vector was isolated by treating with restriction enzyme Ndel 37° C. for 1 hour, it was subjected to a 0.8% agarose gel electrophoresis.

As shown in FIG. 3 b, the DNA fragments of about 0.5 kb for the full-length forms and vector fragments of about 3 kb were detected, confirming that the insert DNA of the CP-Nm23 recombinant construct was appropriately subcloned into the pGEM-T Easy vector.

A pET-28(+)a vector (NOVAGEN, Madison, Wis.) bearing a histidine-tag and a T7 promoter was digested with a restriction enzyme Ndel ( ENZYNOMICS, Korea). The pGEM-T Easy vector fragments containing the CP-Nm23 recombinant fragment and pET-28(+)a vector fragment were purified by using a QIAquick™ Gel extraction kit. Each of the pGEM-T Easy vector fragments was cloned into the pre-treated pET-28a(+) with a T4 ligase at 16° C. for 12 hours, followed by transformation of E. coli DH5α competent cells with the resulting pET-28a(+) vector (FIG. 4 a).

After the clones were treated with the restriction enzyme NdeI (Enzynomics, Korea) and subjected to 0.8% agarose gel electrophoresis, it was verified that DNA fragments of about 0.5 kb for the full-length forms and vector fragments of about 5 kb were detected, confirming the cloning of the insert DNA of CP-Nm23 recombinant construct into pET-28a(+) vector, as shown in FIG. 4 b.

The successfully cloned expression vectors for expressing cell permeable Nm23 recombinant proteins were designated pET28a(+)-HM₁N, pET28a(+)-HNM₁, pET28a(+)-HM₁NM₁, pET28a(+)-HM₂N, pET28a(+)-HNM₂, pET28a(+)-HM₃N, pET28a(+)-HNM₃ and pET28a(+)-HM₃NM₃, respectively. Among them, the E. coli transformants DH5α/HM₃Nm23 and DH5α/HNm23M₃ obtained by transforming E. coli DH5α with the expression vectors pET28a(+)-HM₃N and pET28a(+)-HNM₃, respectively, were deposited on Aug. 28, 2008 in accordance with the Budapest Treaty under accession numbers KCTC-11380BP and KCTC-11381BP with the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong, Yusong-Ku, Taejon 305-333, Republic of Korea.

The results of sequencing analysis are as follows:

As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a kFGF4-derived MTD, His-MTD₁-Nm23 (HM₁N) has an amino acid sequence represented by SEQ ID NO: 22, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 21; His-Nm23-MTD₁ (HNM₁) has an amino acid sequence represented by SEQ ID NO: 24, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 23; and His-MTD₁-Nm23-MTD₁ (HM₁NM₁) has an amino acid sequence represented by SEQ ID NO: 26, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 25.

As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a JO-76 MTD, His-MTD₂-Nm23 (HM₂N) has an amino acid sequence represented by SEQ ID NO: 28, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 27; and His-Nm23-MTD₂ (HNM₂) has an amino acid sequence represented by SEQ ID NO: 30, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 29.

As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a JO-77 MTD as described above, His-MTD₃-Nm23 (HM₃N) has an amino acid sequence represented by SEQ ID NO: 32, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 31; His-Nm23-MTD₃ (HNM₃) has an amino acid sequence represented by SEQ ID NO: 34, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 33; and His-MTD₃-Nm23-MTD₃ (HM₃NM₃) has an amino acid sequence represented by SEQ ID NO: 36, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 35.

As a control for the cell permeable Nm23 recombinant proteins, His-Nm23 (HN), where a full-length Nm23 is fused only to a nuclear localization sequence (NLS) derived from SV40 large T antigen and a histidine-tag (His-Tag) without any MTD, was constructed. The control protein has an amino acid sequence represented by SEQ ID NO: 20, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 19.

Example 2 Expression of Recombinant Proteins

<2-1>Selection of Optimal Bacterial Strains

To select the optimal bacterial strain for the expression of cell permeable Nm23 recombinant proteins prepared in Example 1 above, the following experiments were carried out in E. coli BL21(DE3), BL21-Gold(DE3), BL21-CodonPlus(DE3) and BL21-Gold(DE3) pLysS strains (STRATAGENE, USA), all of which contain the LacI promoter.

First, each of the expression vectors pET28a(+)-HM₁N, pET28a(+)-HNM₁, pET28a(+)-HM₁NM₁, and pHN (control) was transformed into E. coli BL21(DE3), BL21-Gold(DE3), BL21-CodonPlus(DE3) and BL21-Gold(DE3) pLysS strains, respectively, according to the heat shock method. Further, each of the expression vectors pET28a(+)-HM₂N, pET28a(+)-HNM₂, pET28a(+)-HM₃N, pET28a(+)-HNM₃ and pET28a(+)-HM₃NM₃ was transformed into E. coli BL21-Gold(DE3) strain, respectively, according to the heat shock method. After the transformation, the cells were cultured in an LB agar plate containing 50 μg/ml of kanamycin. Colonies formed on the plate were grown in 1 in of LB medium at 37° C. overnight, followed by culturing at 37° C. in 100 μl of LB medium with vigorous shaking until the optical density 600 (OD₆₀₀) reached 0.5. IPTG (isopropyl-β-D-thiogalactoside) was then added thereto at a final concentration of 0.7 mM to induce the expression of the CP-Nm23 recombinant proteins. Protein induction was prolonged for 2 hours at 30° C. The E. coli culture solutions were harvested by centrifugation at 4° C., 7,000×g for 20 minutes, resuspended in a lysis buffer (100 mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH 8.0), and subjected to ultrasonication on ice using a sonicator equipped with a probe. The cell lysates were centrifuged at 14,000×g for 15 minutes, so as to separate an insoluble fraction from a soluble fraction. The thus obtained soluble and insoluble fractions of CP-Nm23 recombinant proteins expressed in the E. coli strain with IPTG were loaded on a SDS-PAGE gel.

As shown in FIG. 5, although some cell permeable Nm23 recombinant proteins were expressed at a relatively low level in BL21-Gold (DE3), most cell permeable Nm23 recombinant proteins showed the highest expression level in the strain. According to these results, BL21-Gold (DE3) was selected as the optimal strain for the expression of the cell permeable Nm23 recombinant proteins according to the present invention.

<2-2>Expression of Recombinant Proteins

Each of the expression vectors pET28a(+)-HM₁N, pET28a(+)-HNM₁, pET28a(+)-HM₁NM₁, pET28a(+)-HM₂N, pET28a(+)-HNM₂, pET28a(+)-HM₃N, pET28a(+)-HNM₃ and pET28a(+)-HM₃NM₃ was transformed into E. coli BL21-Gold(DE3), selected as the optimal strain in section <2-1> of Example 2 above, followed by inducing their expression through the addition of 0.7 mM IPTG, according to the same method described in section <2-1> of Example 2. After that, soluble and insoluble fractions of CP-Nm23 recombinant proteins obtained therefrom were loaded on a SDS-PAGE gel.

As shown in FIG. 6, it was confirmed that the cell permeable Nm23 recombinant proteins (19 to 20 kDa) expressed in the host cell were mostly included in the insoluble fraction as an inclusion body, and their expression was significantly increased in the presence of IPTG.

Example 3 Purification and Refolding of Recombinant Proteins

<3-1>Purification of Recombinant Proteins

The inducible expression of cell permeable Nm23 recombinant proteins in an E. coli system leads to the formation of insoluble aggregates, which are known as inclusion bodies. To completely solubilize these inclusion bodies, all of the above expressed proteins were denatured by dissolving them in 8 M urea used as a strong denaturing agent.

First, the BL21 Gold(DE3) strains transformed with each of the expression vectors pET28a(+)-HM₁N, pET28a(+)-HNM₁, pET28a(+)-HM₁NM₁, pET28a(+)-HM₂N, pET28a(+)-HNM₂, pET28a(+)-HM₃N, pET28a(+)-HNM₃, pET28a(+)-HM₃NM₃ and pET28a(+)-HM (control) were cultured in 1 l of an LB medium as described in Example 2. Each culture solution was harvested by centrifugation, gently resuspended in 20 of a lysis buffer (HN and HNM₁: 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0; other CP-Nm23: 100 mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH 8.0) without forming bubbles, and subjected to ultrasonication on ice using a sonicator equipped with a microtip. The cells were intermittently sonicated for 30 seconds, followed by cooling for 10 seconds, while setting the power to 25% of the maximum power. The total sonication time was 5 minutes. The cell lysates were centrifuged at 4° C., 4,000×g for 20 minutes, so as to separate the supernatant and the cellular debris pellet. The supernatant was loaded onto a Ni-NTA agarose resin where nitrilotriacetic acid agarose was charged with nickel (Ni). The Ni-NTA agarose resin was equilibrated with the lysis buffer. The supernatant was allowed to absorb onto the resin by gently shaking (using a rotary shaker) at 4° C. for 8 hours or more. The resin absorbed with the inclusion bodies containing the recombinant protein was centrifuged at 4° C., 1,000×g for 5 minutes, to remove the reaction solution and washed with a washing buffer (HN and HNM₁: 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 6.3; other CP-Nm23: 100 mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH 6.3) five times to remove nonspecific absorbed materials. After washing, the proteins absorbed to the resin were eluted with an elution buffer (HN and HNM₁: 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 4.5; other CP-Nm23: 100 mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH 4.5) with stirring for 2 hours or more under acidic conditions of pH 4.5. The eluted proteins were analyzed with 12% SDS-PAGE gel electrophoresis, stained with Coomassie Brilliant Blue R by gently shaking, and destained with a destaining solution.

According to the results shown in FIG. 6, all of the cell permeable Nm23 recombinant proteins fused to a kFGF4-derived MTD, a JO-76 MTD and a JO-77 MTD, respectively, were detected as a single band corresponding to about 19 to 20 kDa, which confirms that the cell permeable Nm23 recombinant proteins of the present invention have been purified from the insoluble fraction.

<3-2>Refolding of Recombinant Proteins

Since the cell permeable Nm23 recombinant proteins of the present invention purified from the insoluble fraction as described in section <3-1> of Example 3 above were denatured by a strong denaturing agent, such as 8 M urea, the denatured proteins must be converted into an active form by a refolding process, as follows.

First, the purified recombinant proteins were subjected to a refolding process by dialyzing them against a refolding buffer (0.55 M guanidine HCl, 0.88 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB, 1 mM glutathione oxidized, and 1 mM glutathione reduced) at 4° C. for 24 hours, thereby removing the denaturing agent. All of the refolded recombinant proteins were dialyzed against a cell culture medium DMEM (Dulbecco's Modified Eagle Medium) by using a dialysis bag (Snakeskin pleated, PIERCE) at 4° C. for 10 hours while stirring. The medium was replaced with fresh DMEM every 3 hours. The cell permeable Nm23 recombinant proteins of the present invention converted into their active form through the refolding process were used in the following experiments.

Example 4 Quantitative Cell Permeability Analysis of Nm23 Recombinant Proteins

In order to quantitatively determine the cell permeability of the cell permeable Nm23 recombinant proteins according to the present invention, the introduction of the proteins into the cell was analyzed by FACS (fluorescence-activated cell sorting) in an animal model, as follows.

The cell permeable Nm23 recombinant proteins refolded into their active form in section <3-2> of Example 3 above were labeled with FITC (fluorescein-5-isothiocyanate, MOLECULAR PROBE). The recombinant protein (2 to 20 mg) was mixed with 1 μl of FITC at a concentration of 333 mg/ml and reacted in a dark room at room temperature for 1 hour with gentle stirring. The reaction solution was subjected to a dialysis against DMEM at 4° C. for 2 days until the unreacted FITC was completely removed, thereby obtaining FITC-conjugated recombinant proteins. Thus obtained FITC-conjugated recombinant proteins were subjected to a Bradford protein assay to measure the protein concentration. As a result, each of the FITC-conjugated recombinant proteins was measured to have a concentration of about 0.7 μg/μl.

Meanwhile, RAW 264.7 cells derived from mouse macrophage were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (500 mg/ml) and incubated at 37° C. in a humidified atmosphere of 5% CO₂ in air.

After the incubation, the cells were treated with 10 μM of each of the FITC-conjugated recombinant proteins (HM₁N, HNM₁, HM₁NM₁, HM₃N, HNM₃ and HM₃NM₃) prepared above, followed by further culturing them for 1 hour at 37° C. Subsequently, the cells were treated with trypsin/EDTA (T/E, INVITROGEN) to remove cell surface bound proteins, washed with cold PBS (phosphate buffered saline) three times, and then, subjected to flow cytometry analysis by using a CELLQUEST PRO software program of the FACS (fluorescence-activated cell sorting) Calibur system ( BECKTON-DICKINSON). The cell concentration of each sample was 1×10⁴ cells/μl, and the analysis was performed twice or more. The cell permeability of the cell permeable Nm23 recombinant proteins according to the present invention was determined by comparing it to that of the control protein (HN) not fused to a MTD.

FIGS. 7 a and 7 b show the results of the flow cytometry analysis where the gray filled curve represents cell only, the black curve represents FITC only, the blue curve represents the cell permeability of the control protein not fused to a MTD (HN), the red curve represents the cell permeability of the cell permeable recombinant proteins HM₁N, HM₃N, HNM₃ and HM₃NM₃, the green curve represents the cell permeability of the cell permeable recombinant protein HNM₁, and the orange curve represents the cell permeability of the cell permeable recombinant protein HM₁NM₁. Referring to the results shown in FIGS. 7 a and 7 b, it was found that all of the cell permeable Nm23 recombinant proteins showed significantly higher cell permeability than the control protein.

Example 5 Microscopic Cell Permeability Analysis of Nm23 Recombinant Proteins

To visualize the intracellular localization of human Nm23 proteins delivered into a cell, NIH 3T3 cells (Korean Cell Line Bank, Seoul, Republic of Korea) were treated with FITC-conjugated recombinant proteins (HM₁N, HNM₁, HM₁NM₁, HM₂N, HNM₂, HM₃N, HNM₃ and HM₃NM₃) and visualized by confocal laser scanning microscopy.

First, the NIH 3T3 cells were cultured in an 8-well chamber slide (LabTek, Nalgen Nunc) for 24 hours. The NIH3T3 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 5% penicillin/streptomycin (500 mg/ml) in 5% CO₂ at 37° C. After the cells were washed with PBS three times, the cells were treated with serum-free DMEM, serum-free DMEM containing FITC, and serum-free DMEM containing 10 μM of each of FITC-conjugated recombinant proteins, respectively, in 5% CO₂ at 37° C. One hour later, the cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes.

For a direct detection of FITC-conjugated recombinant proteins that were internalized, the cells were washed with PBS three times and counterstained with a nuclear fluorescent stain solution, propidium iodide (PI, SIGMA-ALDRICH). The cells were stained with PI at a concentration of 1 μg/ml for 5 minutes, followed by washing with PBS three times. In order to preserve the FITC fluorescence of the recombinant protein, the glass slide was fixed in 10 μl of a polyvinyl alcohol mounting medium containing DABCO (Fluca) for 15 minutes before the observation. The intracellular distribution of the fluorescence was determined at the middle of a single cell analyzed by a confocal laser scanning microscope using a normaski filter. The confocal laser scanning microscopy was employed for observing cell phormology, FITC fluorescence and PI fluorescence. FITC was excited at 488 nm and detected by means of a bandpass filter at 530 nm.

As shown in FIGS. 8 a to 8 c, it was observed that the cell permeable Nm23 recombinant proteins stained with FITC and PI were well distributed largely in the nucleus as compared with the cell only, FITC only and MTD-lacking control protein. The intracellular localization of the cell permeable Nm23 recombinant proteins fused to one of a kFGF4-derived MTD, a JO-76 MTD and a JO-77 MTD according to the present invention was consistent with the cell permeability of the same determined by flow cytometry above. From these results, it was confirmed that the cell permeable Nm23 recombinant proteins of the present invention exhibited high cell permeability.

Example 6 Inhibitory Effect of Cell Permeable Nm23 Recombinant Proteins on MAPK Signal Transduction

In order to confirm the in vivo function of the cell permeable Nm23 recombinant proteins according to the present invention, the biochemical functions of the recombinant proteins were examined on three types of cancer cell lines by Western blot analysis.

MDA-MB-435 and MDA-MB-231 cells, the highly metastatic human breast cancer cell lines used in this experiment, were purchased from Korean Cell Line Bank (Seoul, Republic of Korea). The cell lines were maintained in a RPMI 1640 medium (L-glutamine 300 mg/l, 25 mM HEPES and 25 mM NaHCO₃) supplemented with 10% FBS and 1% penicillin/streptomycin in a 5% CO₂ incubator at 37° C. CCL-185 cells, a human lung cancer cell line, were obtained from ATCC and maintained in a HamF-12K medium (2 mM L-glutamine, 1500 mg/f sodium bicarbonate) supplemented with 10% FBS and 1% penicillin/streptomycin in a 5% CO₂ incubator at 37° C.

After 2 ml of the RPMI 1640 medium supplemented with FBS was added to each well of a 6-well plate, MDA-MB-435, MDA-MB-231, and CCL-185 cells were inoculated thereto at a concentration of 5×10⁶ cells/ml. The well plate was incubated at 37° C. for 1 day so as to allow the cells to grow while adhering to the well plate. After removing the medium, the cells adhered to the well plate were washed with cold PBS. Subsequently, the cells were treated with 500 μl of each of the cell permeable Nm23 recombinant proteins and MTD-lacking Nm23 control protein (HIN) at a concentration of 10 μM, and reacted in a 5% CO₂ incubator at 37° C. for 1 hour. The MDA-MB-435 cells were treated with each of HM₁N, HNM₁, HM₁NM₁, HM₂N, HNM₂, HM₃N, HNM₃ and HM₃NM₃ recombinant proteins, while MDA-MB-231 and CCL-185 cells were treated with each of HM₃N, HNM₃ and HM₃NM₃ recombinant proteins. After the reaction was completed, the cells were washed twice with PBS, and then, cultured in the presence of serum under the same conditions noted above for 2, 4, 6 and 8 hours, respectively.

After the cultivation was completed, the cells were resuspended in 200 μl of a lysis buffer (20 mM HEPES, pH 7.2, 1% Triton-X, 10% glycerol and proteinase inhibitor) and subjected to ultrasonication on ice for 30 minutes, to thereby obtain a cell lysate. The cell lysate was centrifuged at 12,000 rpm for 20 minutes at 4° C. to separate the supernatant. The thus obtained supernatant was subjected to a Bradford protein assay to quantitatively measure the protein concentration. The recombinant protein was resuspended in a SDS-PAGE loading buffer at a concentration of 25 μM to prepare a cell lysate sample. The thus prepared cell lysate sample was heated at 90° C. for 5 minutes, and then, stored at −80° C. until use.

For the Western blot analysis, p21 (21 kDa, CELL SIGNALING TECHNOLOGY), phospho-p53 (Ser15, 53 kDa, Cell Signaling), phospho-MEK (Ser217/221, 45 kDa, CELL SIGNALING TECHNOLOGY), and phospho-Erk (Thr202/Tyr204, 42/44 kDa, CELL SIGNALING TECHNOLOGY) were used as primary antibodies, and goat anti-mouse IgG-HRP ( SANTA CRUZ BIOTECHNOLOGY) and goat anti-rabbit IgG-HRP (SANTA CRUZ BIOTECHNOLOGY) were used as secondary antibodies. The cell lysate sample was applied to a 12% SDS-PAGE at 100 V for 2 hours and transferred onto a polyvinylidene fluoride (PDVF) membrane at 100 V for 90 minutes. In order to prevent the nonspecific interaction between the blotted proteins and unrelated antibodies, the PVDF membrane was blocked with 5% non-fat dry milk in TBS/T (10 Mm Tris-Cl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) at room temperature for 1 hour. After removing the blocking buffer, the PVDF membrane was washed with TBS/T, followed by incubation with each of the primary antibodies (diluted with the freshly prepared blocking buffer at a ratio of 1:10000) for 1 hour at 4° C. After removing the primary antibody solution, the membrane was washed with TBS/T five times each for 5 minutes, and incubated with the secondary antibody (diluted with the freshly prepared blocking buffer at a ratio of 1:5000) for 1 hour at room temperature. After washing with TBS/T five times, the membrane was stained using an enhanced chemiluminescence (ECL) detection system (GE HEALTHCARE AMERSHAM UK) to visualize the antigen/antibody interaction.

As shown in FIGS. 9 a and 9 b, in the cells treated with the cell permeable Nm23 recombinant protein as compared with cells treated with the control protein, the phosphorylation of KSR serine 392, which is a scaffold protein of the MAPK cascade, was enhanced, while the phosphorylation of MEK (P-MEK) that induces the activation of tumor cell cycle was reduced. In particular, the HM₃N and HNM₃ recombinant proteins where a JO-77 MTD was fused to the N- and C-terminus, respectively, strongly inhibited the phosphorylation of ERK and MEK in all three types of human cancer cell lines.

Example 7 In Vitro Anti-Metastatic Effect of Cell Permeable Nm23 Recombinant Proteins

<7-1>Invasion assay

In order to examine whether tumor metastasis is inhibited by blocking cancer cell migration in cancer cells treated with the cell permeable Nm23 recombinant proteins according to the present invention, an invasion assay was carried out as follows.

First, a human breast cancer cell line, MDA-MB-435 cells, were cultured overnight in a RPMI 1640 medium supplemented with 10% FBS in the absence of growth factors. The next day, the cells were treated with trypsin and harvested, followed by suspension in the same RPMI 1640 medium. The cells were treated with each of the MTD-lacking Nm23 control protein (HN), and cell permeable Nm23 recombinant proteins (HM₂N, HNM₂, HM₃N, HNM₃ and HM₃NM₃) according to the present invention at a concentration of 10 μM at 37° C. for 1 hour. Meanwhile, the top part of a trans-well polycarbonate membrane filter (BD Falcon) having a pore size of 3 μm was coated with MATRIGEL (40 μg per each well; BD Biosciences). To the lower part of the chamber, a DMEM medium supplemented with 10% FBS was added as an adhesive substrate. The cells treated with the above protein were suspended in a DMEM medium supplemented with 0.1% FBS to prepare a cell suspension. The thus prepared cell suspension was inoculated on the trans-well membrane filter (1×10⁵ cells per each well), and cultured in a 5% CO₂ incubator at 37° C. for 20 to 24 hours. The filters were washed with PBS, and the non-invasive cells remaining on the surface of the upper part were removed by using a cotton swab. The invasive cells that passed through the Matrigel and migrated to the lower part of the filter were fixed with 4% paraformaldehyde for 5 to 10 minutes, and stained with 0.5% (w/v) hemacolor for 10 to 20 minutes. The number of cells migrated to the base surface of the membrane filter (violet color) was counted by observing with an optical microscope.

According to the results shown in FIGS. 10 a and 10 b, in case of the cells treated with most of the cell permeable Nm23 recombinant proteins, HM₂N, HM₃N and HNM₃ in particular, as compared with the control protein (HN), the invasion of the cells was significantly reduced. From these results, it was found that the cell permeable Nm23 recombinant proteins according to the present invention can effectively inhibit metastatic potential of tumor cells in vivo.

<7-2>Wound migration assay

In order to examine whether the cell permeable Nm23 recombinant proteins according to the present invention can inhibit the migration of a breast cancer cell line, MDA-MB-435 cells, having high migration activity, a wound migration assay was carried out as follows.

MDA-MB-435 cells were cultured in a 60-mm culture dish until they formed a confluent monolayer covering the bottom thereof. After incubation, the cells were treated with each of the MTD-lacking Nm23 control protein (HN) and cell permeable Nm23 recombinant proteins (HM₂N, HNM₂, HM₃N, HNM₃ and HM₃NM₃) according to the present invention at a concentration of 10 μM at 37° C. for 1 hour. After the cells were washed with PBS, they were wounded with a sterile yellow tip, to thereby form a reference line that separated the confluent area from the bare area. To the cells was added a RPMI medium (3 mL) supplemented with 10% FBS, followed by culturing in a 5% CO₂ incubator at 37° C. for 24 hours. The cells were washed with PBS, fixed with methanol for 1 minute, stained with Giemsa (Chameleon Chemical) for 5 minutes, and then, washed with distilled water. The migration was quantified by counting the number of cells that migrated from the wound edge into the bare area with an inverted light microscope at 40× magnification.

Referring to the results shown in FIG. 11, the migration of tumor cells was remarkably inhibited in the cells treated with the cell permeable Nm23 recombinant proteins, HM₂N, HM₃N and HNM₃ in particular, as compared with the control protein, which is consistent with the results from the invasion assay above.

Example 8 In Vivo Anti-Metastatic Effect of Cell Permeable Nm23 Recombinant Proteins

In order to examine the in vivo inhibitory effect of the cell permeable Nm23 recombinant proteins on tumor metastasis which has already been confirmed in vitro, an immunohistochemical analysis was carried out as follows.

First, MDA-MB-435 cells, a highly metastatic human breast cancer cell line, were suspended in 0.1 of PBS at a concentration of 1×10⁶ cells/ml and were injected to the outer tail vein of 5-week old MHC (major histocompatibility complex)-deficient Balb/c nu/nu mice. Twenty mice were subdivided into 4 groups of 5 mice each. Each of the cell permeable Nm23 recombinant protein (HM₃N, 300 μg) to which a JO-77 MTD was fused, a vehicle (PBS, 300 μg) and MTD-lacking Nm23 control protein (HN, 300 μg), and an EGFP recombinant protein (HM₃E) where a JO-77 MTD was fused to the N-terminus of EGFP was administered to the mice. Here, the MTD-fused EGFP recombinant protein was employed as a control to examine whether the JO-77 MTD being fused to Nm23 had an effect on Nm23 expression. Five weeks after MDA-MB-435 cells were injected to the mice, the proteins were administered daily to the mice of each group via intravenous injection for 21 days. After three mice were selected from each group and sacrificed, lung tissue samples were extracted therefrom. The other two mice remaining in each group had undergone further observation for 14 days after the administration was terminated, and then, lung tissue samples were extracted therefrom. The lung tissue samples were fixed with a Bouin fixation solution overnight for detecting metastatic colonies, washed with distilled water, and then embedded in paraffin to prepare a paraffin block. Thus prepared paraffin block was sliced with a microtome to have a thickness of 4 μm, where the slices were mounted on a glass slide and treated with xylene for 5 minutes three times to remove paraffin. The glass slide was subjected to immunohistochemical staining with vimentin as a metastatic marker.

For the immunohistochemical staining, anti-vimentin antibody (ABCAM) was employed as a primary antibody, and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) was used as a secondary antibody. In order to prevent the nonspecific interaction between blotted proteins and irrelevant antibodies, the glass slide was blocked with 5% non-fat dry milk in TBS/T (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) at room temperature for 1 hour with stirring. After removing the blocking buffer, the glass slide was washed with TBS/T three times, followed by incubating with the anti-vimentin antibody as a primary antibody (diluted with PBS at a ratio of 1:200) for 1 hour at 4° C. After the removing the primary antibody solution, the glass slide was washed with TBS/T five times each for 5 minutes, and incubated with the goat anti-mouse IgG-HRP as a secondary antibody (diluted with PBS at a ratio of 1:200) for 1 hour at room temperature. After washing with TBS/T (0.025% Triton-X 100) twice, the glass slide was stained with a DAB substrate to detect vimentin.

FIG. 12 a shows the results of optically observing the lung tissue extracted from the mouse after the cell permeable Nm23 recombinant protein according to the present invention was administered for 21 days, and the same was extracted from the mouse where the administration of the cell permeable Nm23 recombinant protein was terminated for 14 days. As shown in FIG. 12 a, in the lung tissues of mice treated with the vehicle, control protein (HN), and MTD-fused EGFP recombinant protein (HM₃E), tumor growth was remarkably increased in spite of the treatment with the proteins for 21 days. Further, the tumor size was not reduced after the subsequent non-treatment period of 2 weeks and newly formed tumors were found in other peripheral tissues, showing that metastasis occurred. However, in the lung tissue of the mouse treated with the cell permeable Nm23 recombinant protein (HM₃N) according to the present invention, there was no tumor formation not only during the 3-week period of protein treatment, but also during the subsequent 2-week period of the non-treatment, suggesting that the tumor formation and metastasis are effectively inhibited by the cell permeable Nm23 recombinant protein.

FIG. 12 b depicts the results of immunohistochemical staining showing the expression of a metastatic marker, vimentin, in the lung tissue extracted from the mouse after the cell permeable Nm23 recombinant protein according to the present invention was administered for 21 days (Day 21), and the lung tissue extracted from the mouse where the administration of the cell permeable Nm23 recombinant protein was terminated for 14 days (Day 35). As shown in FIG. 12 b, vimentin was detected in the lung tissue of the mice treated with the vehicle, control protein (HN), and MTD-fused EGFP recombinant protein (HM₃E) both on Day 21 and on Day 35, while vimentin was not detected in the lung tissue of the mouse treated with the cell permeable Nm23 recombinant protein (HM₃N) according to the present invention neither on Day 21 nor on Day 35. From these results, it was confirmed that the cell permeable Nm23 recombinant protein according to the present invention can effectively inhibit tumor metastasis in vivo.

Example 9 In Vivo Apoptosis-Inducing Effect after the Administration of Cell Permeable Nm23 Recombinant Proteins

In order to examine the effect of inducing apoptosis in tumor tissues after the administration of the cell permeable Nm23 recombinant proteins, a TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay was performed by using the same mouse model as described in Example 8. The TUNEL assay was carried out by using an in situ cell death detection kit (TMR red, ROCHE).

In particular, each of the cell permeable Nm23 recombinant protein (HM₃N), vehicle and HN as a control, and MTD-fused EGFE recombinant protein (HM₃E) was daily administered to the mice subdivided into four groups via intravenous injection for 21 days according to the same method as described in Example 8. After three mice were selected from each group and sacrificed, lung tissue samples were extracted therefrom. The other two mice remaining in each group had undergone further observation for 14 days after the administration was terminated, and then, lung tissue samples were extracted therefrom. The lung tissue samples were embedded in paraffin to prepare a paraffin block. Thus prepared paraffin block was sectioned with a microtome to have a thickness of 5 μm and mounted on a glass slide. The glass slide was treated with xylene for 5 minutes three times, to thereby remove paraffin. It was then successively treated with 100% ethanol twice for 5 minutes, and 90%, 80% and 70% ethanol each for 3 minutes so as to dehydrate the lung tissue, followed by incubation in PBS for 5 minutes. The glass slide was treated with 0.1% Trition® X-100 dissolved in a 0.1% sodium citrate solution for 8 minutes, and washed with PBS twice for 2 minutes. After a drop of TUNEL reaction buffer (50 μl, ROCHE, USA) was added to the glass slide, the glass slide was incubated in a humidified incubator at 37° C. for 1 hour, washed with PBS three times, and then, observed with a fluorescence microscope.

Referring to the results shown in FIG. 13, there was no significant histological change in the mouse lung tissue treated with the vehicle, control protein (FIN) and MTD-fused EGFP recombinant protein, while in the mouse lung tissue treated with the cell permeable Nm23 recombinant protein (HM₃N), a region stained in red representing the characteristic of apoptosis was observed, confirming the effect of inducing apoptosis of the cell permeable Nm23 recombinant protein according to the present invention. Further, it was also observed that in the mouse lung tissue treated with the cell permeable Nm23 recombinant protein according to the present invention, apoptosis was still induced in cancer cells 14 days after the administration was terminated.

Example 10 Comparison of Protein Expression Pattern after the Administration of Cell Permeable Nm23 Recombinant Proteins

In order to examine the change in protein expression pattern in the tumor tissue treated with the cell permeable Nm23 recombinant protein according to the present invention, a microarray assay was performed as follows.

In particular, each of the cell permeable Nm23 recombinant protein (HM₃N), vehicle and HN (control) was administered to the mice subdivided into three groups via intravenous injection for 21 days, and then left alone for 14 days after the administration was terminated, according to the same method as described in Example 9 above. Fourteen days after the administration was terminated, lung tissue samples were extracted from the mouse of each group and freezed with liquid nitrogen. Total RNA was isolated from the lung tissue by using a TRIZOL reagent (INVITROGEN) according to the manufacturer's instruction, and treated with an RNase-free DNase (LIFE TECHNOLOGIES, Inc.), to thereby completely remove the remaining genomic DNA.

The thus isolated RNA was subjected to synthesis and hybridization of a target cRNA probe by using a Low RNA Input Linear Amplification kit (Agilent Technology) according to the manufacturer's instruction. In brief, 1 μg of total RNA was mixed with a T7 promoter specific primer and reacted at 65° C. for 10 minutes. A cDNA master mix was prepared by mixing a first strand buffer (5×), 0.1 M DTT, 10 mM dNTP mix, RNase-Out and MMLV-RT (reverse transcriptase), and added to the reaction mixture. The resulting mixture was reacted at 40° C. for 2 hours, followed by reacting at 65° C. for 15 minutes, to thereby terminate the reverse transcription and dsDNA synthesis. A transcription master mix was prepared by mixing a transcription buffer (4×), 0.1 M DTT, NTP mix, 50% PEG, RNase-Out, inorganic pyrophosphatase, T7-RNA polymerase and cyanine (3/5-CTP) according to the manufacturer's instruction. The thus prepared transcription master mix was added to the dsDNA reaction mixture and reacted at 40° C. for 2 hours so as to perform dsDNA transcription. The thus amplified and labeled cRNA was purified with a cRNA Cleanup Module (AGILENT TECHNOLOGY) according to the manufacturer's instruction. The labeled target cRNA was quantified by using a ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). After the labeling efficiency was examined, cRNA was mixed with a blocking agent (10×) and a fragmentation buffer (25×), and reacted at 60° C. for 30 minutes so as to carry out the fragmentation of cRNA. The fragmented cRNA was resuspended in a hybridization buffer (2×) and directly dropped on a Whole Human Genome Oligo Microarray (44K). The microarray was subjected to hybridization in a hybridization oven (Agilent Technology) at 65° C. for 17 hours, followed by washing according to the manufacturer's instruction (AGILENT Technology).

The hybridization pattern was read by using a DNA microarray scanner ( AGILENT Technology) and quantified by using a Feature Extraction Software ( AGILENT Technology). Data normalization and selection of fold-changed genes were carried out by using a Gene Spring GX 7.3 soft wear (AGILENT Technology). The average of the normalized ratio was calculated by dividing a normalized signal channel strength by a normalized control channel strength. Functional annotation for a gene was conducted by using a Gene Spring GX 7.3 software (AGILENT Technology) according to the Gene Ontology™ Consortium.

The results of the microarray analysis are summarized in FIG. 14 and Tables 3 to 9, where Table 3 shows the expression pattern of apoptosis-relating genes, Table 4 shows that of cell adhesion-relating genes, Table 5 shows that of cell cycle regulation-relating genes, Tables 6a and 6b show that of cell growth-relating genes, Table 7 shows that of cell proliferation-relating genes, Tables 8a and 8b show that of immue response-relating genes, and Table 9 shows that of metastasis-relating genes.

TABLE 3 Exp. pattern Total Veh. vs Veh. vs relative t-test/ Gene Genbank ID Nm23 CP-Nm23 ratio p-value Caspase 14 NM_009809 1.21 4.19 3.46 0.82/0.01  CD5 antigen NM_007650 1.21 3.06 2.53 0.20/0.006 PERP, TP53 apoptosis NM_022032 1.04 2.51 2.41 0.72/0.01  effector CD28 antigen NM_007642 0.81 2.70 3.32 0.29/7.79E−14 ELL associated factor 2 NM_134111 0.72 3.85 5.33 0.08/0.009 Interferon gamma NM_008337 0.76 5.73 7.55 0.33/0.006 Lectin, galactose NM_008496 0.79 8.83 11.16 0.33/0.006 binding, soluble 7 Programmed cell death 1 NM_008798 0.98 4.74 4.85 0.85/0.008 Transformation related NM_011641 2.61 8.22 3.15 0.04/0.009 protein 63 Deoxyribonuclease 1-like 3 NM_007870 1.83 7.37 4.03 0.023/0.006  Fas apoptotic inhibitory NM_026976 0.47 1.40 2.96 0.02/0.08  molecule 3 Interleukin 7 NM_008371 0.82 2.36 2.86 0.028/0.24  Granzyme B NM_013542 0.28 0.72 2.52 0.014/0.09  Cell death-inducing NM_178373 0.64 2.70 4.21 0.01/0.05  DFFA-like effector c Cell death-inducing DNA NM_007702 0.88 2.43 2.77 0.016/0.31  fragmentation factor, alpha subunit-like effector A

TABLE 4 Exp. pattern Total Veh. vs Veh. vs relative t-test/ Gene Genbank ID Nm23 CP-Nm23 ratio p-value Glycosylation dependent cell NM_008134 0.92 4.48 4.88 0.82/0.04 adhesion molecule 1 Selectin, platelet NM_011347 0.94 3.79 4.04 0.83/0.01 CD226 antigen NM_178687 0.89 2.57 2.89 0.50/0.01 Mucin 4 NM_080457 0.93 2.05 2.19 0.58/0.02 Cell adhesion molecule with NM_007697 1.26 5.70 4.54  0.21/0.002 homology to L1CAM RGM domain family, member B BC030405 0.67 3.41 5.11 0.33/0.01 Leupaxin NM_134152 0.76 2.11 2.80  0.1/0.02 Integrin, alpha E, epithelial- NM_008399 0.82 2.66 3.24 0.39/0.02 associated Coagulation factor VIII NM_007977 0.85 2.08 2.43 0.47/0.03 Corneodesmosin NM_001008424 1.87 7.66 4.09  0.21/0.006 Plakophilin 1 NM_019645 8.26 28.39 3.44  0.02/0.005 Stabilin 2 NM_138673 0.40 0.80 1.98 0.038/0.221 Myeloid/lymphoid or mixed AK016557 0.30 0.64 2.12 0.017/0.071 lineage-leukemia translocation to 4 homolog Kit ligand NM_013598 0.20 0.47 2.38 0.008/0.013 Glycoprotein (transmembrane) NM_053110 0.39 1.03 2.64 0.018/0.814 nmb Hairy and enhancer of split 1 NM_008235 0.44 0.98 2.23 0.022/0.910 CD164 antigen NM_016898 0.33 0.89 2.74 0.012/0.377 Procollagen, type XVII, alpha 1 NM_007732 0.36 1.19 3.26 0.017/0.243 Cysteine rich protein 61 NM_010516 0.42 1.14 2.68 0.017/0.327 Cartilage oligomeric matrix NM_016685 3.53 0.66 0.19 0.012/0.127 protein Cadherin-like 26 NM_198656 2.85 0.85 0.30 0.017/0.298 Aggrecan NM_007424 2.84 0.97 0.34 0.013/0.787 Poliovirus receptor-related 4 NM_027893 2.03 0.97 0.48 0.023/0.758 calsyntenin 1 NM_023051 2.17 1.03 0.47 0.020/0.802 laminin, beta 2 NM_008483 2.10 1.05 0.50 0.022/0.665 dermatopontin NM_019759 3.11 1.22 0.39 0.012/0.187 EGF-like repeats and discoidin NM_010103 0.83 0.37 0.45 0.522/0.039 I-like domains 3

TABLE 5 Exp. pattern Total vs vs relative t-test/ Gene Genbank ID Nm23 CP-Nm23 ratio p-value Cell division cycle NM_026560 0.42 2.63 6.23 0.265/0.043 associated 8 Cyclin B1 NM_172301 0.50 2.23 4.46 0.143/0.031 Dedicator of cytokinesis 4 NM_172803 0.66 2.71 4.13 0.174/0.018 Budding uninhibited by NM_009773 0.73 2.95 4.07  0.2/0.016 benzimidazoles 1 homolog, beta Dual specificity phosphatase 1 NM_013642 0.76 2.58 3.39 0.113/0.015 Cell division cycle NM_013538 0.68 2.23 3.27 0.071/0.019 associated 3 FBJ osteosarcoma oncogene NM_010234 0.98 3.44 3.50 0/0 Protamine 1 NM_013637 0.96 2.19 2.28 0.915/0.027 FBJ osteosarcoma oncogene B NM_008036 0.98 3.44 3.50 0.881/0.011 Interleukin 1 beta NM_008361 0.91 2.22 2.45 0.063/0    M-phase phosphoprotein 1 XM_193936 0.40 0.84 2.09  0.03/0.258 Bridging integrator 1 NM_009668 0.29 1.02 3.47 0.012/0.866 Jun-B oncogene NM_008416 0.43 1.00 2.34 0.017/0.977 Calmodulin 1 NM_009790 0.30 1.22 4.11  0.01/0.189 Stratifin NM_018754 2.68 1.03 0.38 0.014/0.883 Avian erythroblastosis virus NM_133659 2.10 0.49 0.23 0.022/0.023 E-26 (v-ets) oncogene related

TABLE 6a Exp. pattern Total Veh. vs Veh. vs relative t-test/ Gene Genbank ID Nm23 CP-Nm23 ratio p-value Keratin 5 NM_027011 2.71 8.04 2.96 0.015/0.006 Myosin, heavy polypeptide 8, AK081482 1.45 2.91 2.00  0.2/0.02 skeletal muscle, perinatal Paired box gene 9 NM_011041 1.58 4.49 2.84 0.059/0.009 Troponin T3, skeletal, fast NM_011620 2.74 6.65 2.43 0.007/0.007 Serine (or cysteine) peptidase AK003650 1.04 9.02 8.71  0.26/0.006 inhibitor, clade B, member 3C B-cell leukemia/lymphoma 2 NM_007535 1.08 2.43 2.26 0.538/0.011 related protein A1c OVO homolog-like 1 NM_019935 1.08 2.26 2.09 0.553/0.014 Junctophilin 2 NM_021566 1.54 14.08 9.12 0.115/0.006 Myosin, light polypeptide 1 NM_021285 1.30 2.65 2.05 0.127/0.014 Histocompatibility 2, class II NM_207105 1.23 3.27 2.66 0.101/0.011 antigen A, beta 1 Troponin I, skeletal, slow 1 NM_021467 0.96 2.24 2.33 0.814/0.025 Growth arrest specific 7 NM_008088 0.94 3.02 3.22 0.619/0.012 Tumor necrosis factor (ligand) NM_009403 0.91 2.89 3.16 0.719/0.016 superfamily, member 8 Frizzled homolog 10 NM_175284 0.99 2.31 2.33 0.946/0.020 Chemokine (C—X—C motif) NM_018866 1.02 5.12 5.02 0.854/0.008 ligand 13 RAB27A, member RAS oncogene NM_023635 0.71 2.24 3.15 0.326/0.033 family Tumor necrosis factor, NM_025566 0.74 2.15 2.88 0.122/0.021 alpha-induced protein 8-like 1 RAB32, member RAS oncogene NM_026405 0.71 2.41 3.38  0.08/0.017 family Tumor necrosis factor, NM_009396 0.74 2.37 3.22 0.096/0.017 alpha-induced protein 2

TABLE 6b Tumor necrosis factor receptor NM_011608 0.65 4.42 6.81 0.083/0.009 superfamily, member 17 V-maf musculoaponeurotic NM_010658 0.83 2.03 2.45 0.203/0.023 fibrosarcoma oncogene family, protein B (avian) Interleukin 7 receptor NM_008372 0.82 2.36 2.86 0.195/0.017 Fgfr1 oncogene partner NM_201230 0.49 1.15 2.33 0.031/0.328 Tescalcin NM_021344 0.50 1.16 2.34 0.025/0.272 Burkitt lymphoma receptor 1 NM_007551 0.42 1.93 4.60 0.031/0.031 Protein phosphatase 1, AK082372 0.18 0.40 2.22 0.019/0.184 regulatory (inhibitor) subunit 14c Eyes absent 4 homolog NM_010167 1.07 0.46 0.43 0.721/0.046 Sema domain, transmembrane AK052232 1.12 0.48 0.43 0.367/0.022 domain (TM), and cytoplasmic domain, (semaphorin) 6D Mutated in colorectal cancers AK086823 0.87 0.41 0.47 0.347/0.019 Sema domain, immunoglobulin AK049580 0.95 0.38 0.40 0.713/0.18 domain (Ig), short basic domain, secreted, (semaphorin) 3E Sine oculis-related homeobox 2 NM_011380 4.18 1.60 0.38 0.022/0.094 homolog Early B-cell factor 3 AK220542 4.49 1.70 0.38 0.008/0.049 V-abl Abelson murine leukemia NM_009595 3.90 1.35 0.35 0.021/0.201 viral oncogene 2 (arg, Abelson-related gene) Early B-cell factor 2 NM_010095 2.88 1.30 0.45 0.015/0.152 HtrA serine peptidase 3 NM_030127 3.50 1.41 0.40 0.011/0.089 Troponin T2, cardiac NM_011619 2.33 1.13 0.49 0.018/0.34  Developmental pluripotency NM_025274 2.30 0.85 0.37 0.027/0.414 associated 5 Receptor tyrosine kinase-like NM_013845 2.33 1.06 0.46 0.022/0.654 orphan receptor 1 Phospholipase C, gamma 1 AF027185 3.05 0.99 0.33 0.012/0.951 Deleted in lung and esophageal AK045848 2.34 0.95 0.41 0.018/0.641 cancer 1 Palate, lung, and nasal NM_011126 3.68 0.14 0.04 0.007/0/007 epithelium carcinoma associated

TABLE 7 Exp. pattern Total Veh. vs Veh. vs relative t-test/ Gene Genbank ID Nm23 CP-Nm23 ratio p-value Protein kinase, cAMP NM_008923 0.87 2.02 2.31 0.494/0.049 dependent regulatory, type I beta B-cell translocation gene 1, NM_007569 0.90 2.02 2.25 0.406/0.024 anti-proliferative Fc receptor, IgG, low NM_010187 0.77 2.16 2.82 0.112/0.018 affinity IIb Immunoglobulin heavy chain AJ294737 0.71 2.11 2.97 0.079/0.021 6 (heavy chain of IgM) Neutrophil cytosolic factor 1 NM_010876 1.02 2.35 2.30 0.888/0.019 complement factor B NM_008198 1.12 3.27 2.91 0.365/0.011 B-cell translocation gene 2, NM_007570 0.30 0.68 2.28 0.548/0.02  anti-proliferative Signal transducer and NM_009284 0.48 2.34 4.88  0.04/0.019 activator of transcription 6 Protein tyrosine NM_011210 0.48 1.35 2.79 0.022/0.1  phosphatase, receptor type, C Protein-kinase, NM_028410 0.31 0.83 2.71 0.011/0.266 interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor) Pleckstrin homology domain AK045134 0.70 0.25 0.36  0.08/0.011 containing, family K member 1

TABLE 8a Exp. pattern Veh. Veh. Total Vs Vs CP- Relative t-test/ Gene Genbank ID Nm23 Nm23 ratio p-value Immunoglobulin AF296432 0.86 16.25 18.85 0.905/ heavy chain 0.011 (J558 family) Immunoglobulin BC004786 0.96 15.09 15.69 0.736/ heavy chain complex 0.006 Histocompatibility 2, T NM_010397 1.00 2.87 2.89 0.966/ region locus 22 0.013 Lymphocyte NM_010745 0.99 2.42 2.44 0.936/ antigen 86 0.016 Defensin NM_054074 0.97 2.19 2.26 0.863/ beta 6 0.025 Immunoglobulin NM_152839 0.84 26.49 31.61 0.219/ joining chain 0.005 Histocompatibility NM_010382 0.93 3.65 3.92 0.55/ 2, class II 0.01 antigen E beta Lymphocyte antigen NM_008530 1.04 3.53 3.41 0.784/ 6 complex, locus F 0.01 Thymus cell antigen NM_009382 1.04 2.47 2.38 0.762/ 1, theta 0.016 CD8 antigen, BC030679 0.78 6.62 8.51 0.142/ alpha chain 0.007 Chemokine NM_009917 0.86 2.65 3.09 0.265/ (C - C motif) 0.014 receptor 5 Proteoglycan 2, NM_008920 0.88 3.64 4.13 0.373/ bone marrow 0.01 Histocompatibility NM_010388 0.93 2.68 2.90 0.518/ 2, class 0.014 II, locus Mb2 Defensin beta 3 NM_013756 1.21 5.89 4.89 0.364/ 0.007 Histocompatibility NM_010388 1.08 2.79 2.57 0.53/ 2, class II, 0.013 locus Mb2 Histocompatibility NM_010389 1.06 2.45 2.31 0.647/ 2, O region 0.017 beta locus Histocompatibility NM_010381 1.11 4.12 3.73 0.423/ 2, class 0.009 II antigen E alpha Transporter 1, NM_013683 1.21 3.38 2.80 0.226/ ATP - binding 0.011 cassette, sub - family B (MDR/TAP) Peptidoglycan NM_009402 1.25 2.71 2.17 0.159/ recognition protein 1 0.014 T - cell receptor beta, BC030075 0.60 2.08 3.47 0.148/ variable 13 0.012 Indoleamine - pyrrole NM_008324 0.68 5.91 8.68 0.079/ 2,3 dioxygenase 0.007 Histocompatibility NM_010387 0.83 2.20 2.64 0.214/ 2, class II, locus Mb1 0.019

TABLE 8b CD3 antigen, gamma NM_009850 0.68 5.05 7.46 0.063/0.008 polypeptide CD96 antigen NM_032465 0.79 2.39 3.02 0.148/0.017 Histocompatibility 2, class NM_010378 0.72 2.75 3.82  0.12/0.014 II antigen A, alpha Histocompatibility 2, 0 NM_008206 0.65 2.81 4.31 0.053/0.013 region alpha locus Chemokine (C-X-C motif) NM_009910 0.63 3.25 5.12 0.048/0.008 receptor 3 CD8 antigen, beta chain 1 NM_009858 0.75 2.15 2.86 0.107/0.021 CD86 antigen NM_019388 0.83 2.29 2.77 0.066/0.01  CD3 antigen, delta polypeptide NM_013487 0.56 3.42 6.16 0.031/0.011 Interferon gamma inducible protein 47 NM_008330 0.67 2.23 3.33  0.06/0.019 Immunoglobulin lambda chain, AK008094 0.40 2.85 7.15 0.044/0.015 variable 1 CD180 antigen NM_008533 0.42 2.19 5.22 0.107/0.042 Coiled-coil domain AK087049 1.00 0.44 0.44 0.984/0.019 containing 85A Cathelicidin antimicrobial NM_009921 1.19 0.37 0.31 0.243/0.016 peptide Neutrophilic granule protein NM_008694 1.41 0.36 0.25 0.083/0.007

TABLE 9 Exp. pattern Total Veh. vs Veh. vs relative t-test/ Gene Genbank ID Nm23 CP-Nm23 ratio p-value Fascin homolog 1, actin NM_007984 1.31 3.56 2.72 0.118/0.01  bundling protein Prostaglandin-endoperoxide NM_011198 0.81 2.09 2.59 0.459/0.047 synthase 2 Vascular cell adhesion NM_011693 0.79 1.62 2.05 0.146/0.044 molecule 1

As described in Table 3 above, in case of the apoptosis-relating genes, while the expressions of Caspase 14, cell death-inducing DFFA-like effector c (Cidec), cell death-inducing DNA fragmentation factor and alpha subunit-like effector A (Cidea) were up-regulated by about 3.5-, 4.0-, 2.5- and 2.5-fold, respectively, in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.

As described in Table 4 above, in case of the cell adhesion-relating genes, the expression of cadherin-like 26 was down-regulated by about 3.0-fold in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.

As described in Table 5 above, in case of the cell cycle regulation-relating genes, the expression of Avian erythroblastosis virus E-26 (v-ets) oncogene was down-regulated by about 4.0-fold in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.

As described in Tables 6a and 6b above, in case of the cell growth-relating genes, while the expression of member 17 of a tumor necrosis factor receptor superfamily was up-regulated by about 6.8-fold, the expressions of palate, lung and nasal epithelium carcinoma associated genes were down-regulated by about 26.0-fold in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.

As described in Table 7 above, in case of the cell proliferation-relating genes, the expression of signal transducer and activator of transcription 6 was up-regulated by about 5-fold in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.

As described in Tables 8a and 8b above, in case of immune response-relating genes, the expressions of immunoglobulin heavy chain (J558 family), immunoglobulin heavy chain complex and immunoglobulin joining chain were up-regulated by about 18-, 15- and 30-fold, respectively, in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.

As described in Table 9 above, in case of metastasis-relating genes, the expressions of fascin homolog 1 (actin bundling protein), prostaglandin-endoperoxide synthase 2 and vascular cell adhesion molecule 1 were up-regulated by about 2.5-, 2.5- and 2.0-fold, respectively, in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

INDUSTRIAL APPLICABILITY

The cell permeable Nm23 recombinant proteins of the present invention can induce the KSR phosphorylation and inactivation and inhibit the Ras-mediated MAPK cascade by efficiently introducing a metastasis suppressor Nm23 into a cell. Therefore, the cell permeable Nm23 recombinant proteins of the present invention can be effectively used as an anti-metastatic agent capable of preventing cancer metastasis by suppressing the proliferation, differentiation and migration of cancer cells. 

The invention claimed is:
 1. A cell permeable Nm23 recombinant protein comprising: a macromolecule transduction domain(MTD) fused to the N-terminus or C-terminus of a human metastasis suppressor Nm23, wherein the MTD comprises SEQ ID NO: 6 or 8, and the Nm23 comprises SEQ ID NO:
 2. 2. A cell permeable Nm23 recombinant protein comprising: a first MTD fused to the N-terminus of a human metastasis suppressor Nm23 and a second MTD fused to the C-terminus of said human metastasis suppressor, wherein the first and second MTDs comprise SEQ ID NO: 6 or 8 and the Nm23 comprises SEQ ID NO:
 2. 3. The cell permeable Nm23 recombinant protein according to claim 1, wherein the recombinant protein is selected from the group consisting of: a recombinant protein wherein the MTD comprises SEQ ID NO:6 and is fused to the N-terminus of the Nm23; a recombinant protein wherein the MTD comprises SEQ ID NO:6 and is fused to the C-terminus of the Nm23; a recombinant protein wherein the MTD comprises SEQ ID NO: 8 and is fused to the N-terminus of the Nm23; and a recombinant protein wherein the MTD comprises SEQ ID NO:8 and is fused to the C-terminus of the Nm23.
 4. The cell permeable Nm23 recombinant protein according to claim 1, wherein the recombinant protein has an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 30, 32, and
 34. 5. A polynucleotide encoding the cell permeable Nm23 recombinant protein according to claim
 1. 6. The polynucleotide according to claim 5, wherein the polynucleotide has a nucleotide sequence selected form the group consisting of SEQ ID NOS: 27, 29, 31, and
 33. 7. An expression vector comprising the polynucleotide according to claim
 5. 8. The expression vector according to claim 7, wherein the expression vector is selected from the group consisting of pET28a(+)-HM₂N, pET28a(+)-HNM₂, and pET28(+)-HM₃N, pET28a(+) -HNM.
 9. A transformant comprising the expression vector according to claim
 7. 10. The transformant according to claim 9, wherein the transformant is E. coli DH5α/HM₃Nm23 (Accession No. KCTC- 11380BP).
 11. The transformant according to claim 9, wherein the transformant is E. coli DH5α/HNm23M₃ (Accession No. KCTC- 11381 BP).
 12. A method of producing a cell permeable Nm23 recombinant protein comprising culturing the transformant according to claim 1 or
 2. 13. The cell permeable Nm23 recombinant protein according to claim 1 or 2, further comprising: a nuclear localization sequence(NLS) and a histidine-tag affinity domain, said nuclear localization sequence and histidine-tag affinity domain being covalently coupled to one end of the recombinant protein.
 14. The cell permeable Nm23 recombinant protein of claim 2 wherein the first and second MTDs comprise SEQ ID NO
 8. 15. The cell permeable Nm23 recombinant protein according to claim 2, wherein the recombinant protein has an amino acid sequence of SEQ ID NO:
 36. 16. A polynucleotide encoding the cell permeable Nm23 recombinant protein according to claim
 2. 17. The polynucleotide according to claim 16, wherein the polynucleotide has a nucleotide sequence of SEQ ID NO:
 35. 18. An expression vector comprising the polynucleotide according to claim
 16. 19. The expression vector according to claim 18, wherein the expression vector is pET28(+)-HM₃NM₃.
 20. A transformant comprising the expression vector according to claim
 18. 21. A pharmaceutical composition comprising the cell permeable Nm23 recombinant protein according to claim 1 or 2 as an effective ingredient and a pharmaceutically acceptable carrier, which if effective for preventing metastasis by inhibiting proliferation, differentiation or migration of cancer cells. 